EP1859026A2 - Differenciation dirigee de cellules souches embryonnaires et utilisations associees - Google Patents

Differenciation dirigee de cellules souches embryonnaires et utilisations associees

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Publication number
EP1859026A2
EP1859026A2 EP06719900A EP06719900A EP1859026A2 EP 1859026 A2 EP1859026 A2 EP 1859026A2 EP 06719900 A EP06719900 A EP 06719900A EP 06719900 A EP06719900 A EP 06719900A EP 1859026 A2 EP1859026 A2 EP 1859026A2
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European Patent Office
Prior art keywords
cells
differentiated
cell
differentiation
days
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EP06719900A
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German (de)
English (en)
Inventor
Alan Colman
William Sun
Norris Ray Dunn
Blaine Phillips
Hannes Martin Hentze
William Lathrop Rust
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ES Cell International Pte Ltd
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ES Cell International Pte Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/148—Transforming growth factor alpha [TGF-a]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • embryonic stem cells Over the last decade, tremendous excitement in the stem cell field has fueled the hope that various stem cell populations will form the basis of treatments for a diverse array of degenerative diseases and disorders. Embryonic stem cells have attracted particular excitement for their seemingly unprecedented ability to differentiate to tissues derived from all three germ layers. Accordingly, embryonic stem cells may form the basis of a wider range of therapeutics than adult stem cells derived from any particular tissue. However, despite the excitement generated by the limitless potential of embryonic stem cells to differentiate along ectodermal, mesodermal, and endodermal lineages, effective therapeutics require the ability to control and direct the differentiation of embryonic stem cells to a particular cell type.
  • the present invention provides methods of promoting the directed differentiation of embryonic stem cells to particular endodermally derived cell types. Such methods can be used to generate cultures of partially and/or terminally differentiated cells that can be used therapeutically to treat or prophylactically treat injuries and diseases of endodermally derived tissues and organs.
  • the invention provides methods for the directed differentiation of cells to endodermal cell types. Endodermal cell types differentiated according to the methods of the present invention can be used to treat or prophylactically treat injuries and diseases of endodermally derived tissues and organs.
  • the present invention provides methods for directing the differentiation of embryonic stem cells to various endodermal cell types. Specifically, the present invention provides methods for directing the differentiation of embryonic stem cells along a pancreatic lineage. In certain embodiments, the methods of the invention lead to the production, from embryonic stem cells, of pdx-l + cells indicative of cells that have begun differentiation to a pancreatic cell fate. In certain other embodiments, the methods of the invention lead to the production, from embryonic stem cells, of insulin producing cells.
  • the methods of the invention lead to the production, from embryonic stem cells, of cells that express insulin and C-peptide, and/or are glucose-responsive.
  • Pdx-1 + cells and/or insulin-producing cells produced using the methods of the present invention can be delivered to human or animal patients and used for the treatment or prophylaxis of conditions of the pancreas.
  • the invention provides a method for directed differentiation of embryonic stem (ES) cells into pancreatic lineage, comprising: contacting the ES cells for a sufficient period of time with a sufficient amount of one or more early factors (EFs) selected from activin A, BMP2, BMP4, or nodal, wherein the pancreatic lineage cells express pancreatic lineage marker(s), and/or exhibit a pancreatic lineage function.
  • EFs early factors
  • the pancreatic lineage cells express Pdx-1 and/or insulin, and/or are responsive to glucose, and/or secret C-peptide.
  • Such pancreatic lineage cells may be Insulin-producing cells, such as pancreatic ⁇ -cells.
  • the ES cells are cultured as embryoid bodies (EBs), plated directly onto a support matrix (such as MATRIGELTM), and/or plated directly onto tissue culture plates.
  • the EBs may be cultured in a floating suspension culture, in a support matrix (such as MATRIGELTM or other matrix), and/or on a filter.
  • Supporting matrices other than MATRIGELTM are known in the art, including basement membrane extractable from placenta as described in Kawaguchi et al., Proc. Natl. Acad. ScL 95(3): 1062-66, 1998; BD Bioscience's PuraMatrix synthetic peptide scaffold; or fibronectin matrix, etc.
  • the EBs are cultured in a support matrix (such as
  • MATRIGELTM only during the period when the EBs are in contact with the EFs.
  • the EBs are generated from ES cells grown on MEF (mouse embryonic feeder) or other feeder layers, or from ES cells grown under feeder-free conditions.
  • MEF mouse embryonic feeder
  • the ES cells are xeno-free, preferably also
  • CGMP- and GTCP-compliant CGMP: Current Good Manufacturing Practice
  • GTCP Good Tissue Culture Practice
  • the ES cells from many different species of animals may be used in the methods of the invention, hi certain embodiments, the ES cells are human ES cells, hi other embodiments, the ES cells are from non-human mammals, such as ES cells from rodents (rats, mice, rabbits, hamsters, etc.); primates (e.g., monkey, apes, etc.), pets (cats, dogs, etc.); livestock animals (cattle, pigs, horses, sheep, goats, etc.).
  • rodents rats, mice, rabbits, hamsters, etc.
  • primates e.g., monkey, apes, etc.
  • pets cats, dogs, etc.
  • livestock animals cattle, pigs, horses, sheep, goats, etc.
  • the human ES cells are from the hES 1, hES2, hES3, hES4, hES5, hES6 or DM ES cell lines.
  • the ES cells are partially or terminally differentiated into the pancreatic lineage.
  • the ES cells are contacted with the EFs for about 15 days, preferably about 10 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or about 14 days.
  • the EFs comprise activin A and BMP4.
  • the EFs comprise about 50 ng/mL (e.g., about 10-200 ng/mL, or about 20-100 ng/mL, or about 30-70 ng/mL, or about 40-60 ng/mL) of activin A and about 50 ng/mL (e.g., about 10-200 ng/mL, or about 20-100 ng/mL, or about 30-70 ng/mL, or about 40-60 ng/mL) of BMP4.
  • ng/mL e.g., about 10-200 ng/mL, or about 20-100 ng/mL, or about 30-70 ng/mL, or about 40-60 ng/mL
  • the method further comprises contacting the ES cells, subsequent to contacting the ES cells with the EFs, with a sufficient amount of one or more late factors (LFs) for a second sufficient period of time.
  • the LFs may be HGF, exendin4, betacellulin, and nicotinamide
  • the one or more LFs include about 50 ng/mL (e.g., about 10-200 ng/mL, or about 20-100 ng/mL, or about 30-70 ng/mL, or about 40-60 ng/mL) of HGF, about 10 ng/mL (e.g., about 2-50 ng/mL, or about 5-20 ng/mL) of exendin4, and about 50 ng/mL (e.g., about 10-200 ng/mL, or about 20-100 ng/mL, or about 30-70 ng/niL, or about 40-60 ng/mL) of ⁇ -cellulin.
  • the ES cells are contacted with the EFs for about 10 days, and are subsequently contacted with the LFs for about 10 days.
  • the EFs comprise about 50 ng/mL of activin A and about 50 ng/mL of BMP4, and the LFs include about 50 ng/mL of HGF, about 10 ng/mL of exendin4, and about 50 ng/mL of ⁇ -cellulin.
  • the method further comprises contacting the ES cells, subsequent to the initiation protocol and during a maturation protocol, consecutively with: (1) a basal medium for about 6 days; (2) about 20 ng/ml (e.g., about 5-100 ng/mL, or about 10-40 ng/mL) FGF-18, and about 2 ⁇ g/ml (e.g., about 0.5-10 ⁇ g/ml, or about 1-5 ⁇ g/ml) Heparin in the basal medium for about 5-6 days; (3) about 20 ng/ml (e.g., about 5-100 ng/mL, or about 10-40 ng/mL) FGF-18, about 2 ⁇ g/ml (e.g., about 0.5-10 ⁇ g/ml, or about 1-5 ⁇ g/ml) Heparin, about 10 ng/ml EGF (e.g., about 2-50 ng/mL, or about 5-20 ng/mL), about 4 ng/ml TGF- ⁇
  • steps (1) - (3) use DMEM/F12 medium or equivalents.
  • step (4) uses RPMI 1640 or equivalent medium.
  • step (5) uses CMRL medium.
  • the ES cells are not dissociated by dispase between step (1) and (2).
  • FBS if any is used
  • FBS in the medium is replaced with a chemically defined serum replacer (SR).
  • the method further comprises contacting the ES cells, subsequent to the EF and LF treatment, and during a maturation protocol, with about 10 ⁇ M (e.g., about 2-50 ⁇ M, or 5-20 ⁇ M) Forskolin, about 40 ng/ml (e.g., about 10-150 ng/mL, or about 20-80 ng/mL) HGF, and about 200 ng/ml (e.g., about 50-800 ng/mL, or about 100-400 ng/mL) PYY for about 3-4 days.
  • 10 ⁇ M e.g., about 2-50 ⁇ M, or 5-20 ⁇ M
  • Forskolin e.g., about 40 ng/ml (e.g., about 10-150 ng/mL, or about 20-80 ng/mL) HGF
  • about 200 ng/ml e.g., about 50-800 ng/mL, or about 100-400 ng/mL
  • the ES cells are grown on fibronectin-coated tissue culture surfaces during the maturation protocol.
  • the differentiated cells release C-peptide and/or are responsive to glucose stimulation.
  • the method further comprises contacting the ES cells, subsequent to contacting the ES cells with the EFs and during a maturation protocol, consecutively with: (1) about 20 ng/ml FGF-18, and about 2 ⁇ g/ml Heparin in a basal medium for about 8 days; (2) about 20 ng/ml FGF-18, about 2 ⁇ g/ml Heparin, about 10 ng/ml EGF, about 4 ng/ml TGF ⁇ , about 30 ng/ml IGFl, about 30 ng/ml IGF2, and about 10 ng/ml VEGF in the basal medium for about 6 days; and (3) about 10 ⁇ M Forskolin, about 40 ng/ml HGF, and about 200 ng/ml PYY for about 5 days. It is contemplated that the range of concentrations of the factors are as those described above.
  • the differentiated cells release C-peptide.
  • step (1) above lasts 6 days, steps (2) and (3) last 4 days each.
  • the invention provides cells and cell clusters differentiated by the methods of the present invention from embryonic stem cells.
  • the cells or cell clusters express pdx-1.
  • the cells or cell clusters express insulin.
  • the cells or cell clusters express and secrete C-peptide.
  • the cells or cell clusters express both insulin and C-peptide.
  • exemplary cells or cell clusters are glucose-responsive.
  • this aspect of the invention provides differentiated pancreatic lineage cells or cell cultures obtained through the various subject methods.
  • the differentiated pancreatic lineage cells or cell cultures are partially differentiated.
  • the differentiated pancreatic lineage cells or cell cultures are terminally differentiated.
  • the differentiated pancreatic lineage cells or cell cultures mimic the function, in whole or in part, of Insulin-producing cells, such as pancreatic beta islet cells.
  • the invention provides methods for the treatment or prophylaxis of diseases, injuries, or conditions of the pancreas.
  • diseases, injuries, or conditions of the pancreas are characterized by impaired pancreatic function, for example, impaired ability to properly regulate glucose metabolism in an affected individual.
  • the disease, injury, or condition of the pancreas is diabetes (e.g., type I or type II diabetes), and the invention provides methods for the treatment or prophylaxis of diabetes, hi one embodiment, the method of treatment comprises administering a composition of partially differentiated cells or cell clusters (e.g.,pdx-l ). In another embodiment, the method of treatment comprises administering a composition of terminally differentiated cells or cells clusters. Such terminally differentiated cells or cell clusters comprises (in whole or in part) glucose responsive cells. In any of the foregoing, the invention contemplates methods of treatment comprising administration of cells or cell clusters differentiated by the methods of the invention along with one or more additional therapies.
  • a composition of partially differentiated cells or cell clusters e.g.,pdx-l
  • the method of treatment comprises administering a composition of terminally differentiated cells or cells clusters.
  • Such terminally differentiated cells or cell clusters comprises (in whole or in part) glucose responsive cells.
  • the invention contemplates methods of treatment comprising administration
  • this aspect of the invention provides a method for the treatment or prophylaxis, in an individual, of diseases, injuries, or conditions of the pancreas characterized by impaired pancreatic function, comprising administering to the individual the subject differentiated pancreatic lineage cells.
  • the impaired pancreatic function includes impaired ability to properly regulate glucose metabolism in an affected individual.
  • the condition is type I or type II diabetes.
  • the method is in conjunction with one or more additional therapies effective for the treatment or prophylaxis of the diseases, injuries, or conditions.
  • the invention provides initiation protocols, maturation protocols, and combinations of initiation and maturation protocols for the directed differentiation of embryonic stem cells along a pancreatic lineage.
  • the initiation protocol, maturation protocol, or combination thereof promotes expression of pdx-1, insulin, and/or C-peptide.
  • the initiation protocol, maturation protocol, or combination thereof promotes induction of glucose responsive cells or cell clusters that mimic the function, in whole or in part, of beta islet cells.
  • the invention contemplates that these methods can be used to direct the differentiation of other adult and fetal stem cell populations to endodermal cell types.
  • Figure 1 shows the results of RT-PCR analysis of human embryonic stem cells allowed to spontaneously differentiate via embryoid body formation. Expression analysis confirmed that human embryonic stem cells can spontaneously differentiate along ectodermal, mesodermal, and endodermal lineages.
  • Figure 2 shows a schematic representation of methods of directing differentiation of a stem cell to a differentiated pancreatic cell.
  • the method proceeds in two stages.
  • the stem cells are directed to differentiate along a particular lineage, for example the pancreatic lineage, by promoting expression of a marker indicative of partial differentiation down a particular lineage.
  • the partially differentiated cells are terminally differentiated to express one or more markers of a particular differentiated cell type.
  • the end point e.g., the goal of a particular differentiation protocol
  • the generation of partially differentiated cells is either generation of partially differentiated cells or the generation of terminally differentiated cells.
  • Figure 3 summarizes the results of experiments in which hES3 were cultured as embryoid bodies suspended in 3D in MATRIGELTM. The cells were cultured for 10 days in medium containing the early factors and then for 10 days in medium containing the late factors. Following culture, cells were assayed for expression of pdx-1. For each bar depicted in Figure 3, the embryoid bodies were cultured, except as indicated, with the following early and late factors: early factors were activin A, BMP2, BMP4, and nodal; late factors were HGF, exendin4, betacellulin, and nicotinamide. The particular factor omitted is indicated under each bar.
  • Figures 4A and 4B show the directed differentiation of a mouse embryonic stem cell along a particular endodermal lineage.
  • FIG. 4A shows a cluster of cells expressing ⁇ -galactosidase (indicating p dx-1 expression) after EB formation and subsequent plating.
  • Figure 4B shows quantitative RT-PCR data for pdx-1 for mouse embryoid bodies at various stages of culture. Pdx-1 expression increased over time up to 24 days of EB formation.
  • Figures 5 A and 5B show that expression of the early pancreatic marker, pdx- 1, increased over time in embryoid bodies formed from human embryonic stem cell line hES2.
  • Figure 5A shows that pdx-1 expression increased between 0 - 24 days of embryoid body formation, as measured by RT-PCR. As a control, actin expression was measured and this expression did not change significantly over time.
  • Figure 5B shows an ethidium bromide stained gel of the pdx-1 RT-PCR product, indicating that a single band of the predicted size was detected.
  • Figure 6 shows that addition of TGF ⁇ family growth factors to embryoid bodies, in culture, increased expression of pdx-1.
  • FIGS. 7A and 7B show the directed differentiation along a particular endodermal lineage.
  • Figure 7A shows a cluster of embryonic stem cells expressing the hepatocyte marker albumin.
  • Figure 7B shows quantitative RT-PCR data examining markers of endodermal differentiation in two different human embryonic stem cell lines undergoing any of several differentiation protocols.
  • Figure 8 shows pdx-1 expression in embryonic stem cells at various time points during culture as embryoid bodies suspended in 3D culture in MATRIGELTM. The cells were cultured for 10 days in medium containing the early factors and then for 10 days in medium containing the late factors.
  • Figures 9A and 9B show expression of pdx-1 and insulin in embryonic stem cells differentiated under a combination of conditions. Cells were cultured as embryoid bodies in 3D cultures for three weeks in the presence of early and late factors, and were then subjected to a 27 day, multi-step differentiation protocol. Figure 9 A shows expression of pdx-1 and Figure 9B shows expression of insulin. Figures 1OA and 1OB show expression of pdx-1 and insulin in embryonic stem cells differentiated under a combination of conditions. Cells were cultured as embryoid bodies in 3D cultures for one week, and were then subjected to a 32 day, multi-step differentiation protocol. Figure 1OA shows expression of pdx-1 and Figure 1OB shows expression of insulin.
  • Figure 11 shows the kinetics of endodermal and pancreatic gene expression during in vitro, directed differentiation of embryonic stem cells.
  • Figure 12 shows a detailed analysis of the temporal pattern of gene expression during in vitro, directed differentiation of embryonic stem cells.
  • the data summarized in Figures 11 and 12 demonstrate that gene expression during the directed differentiation of embryonic stem cells along a pancreatic lineage mimics that which occurs during normal pancreatic development.
  • Figures 13 A and 13B summarize the results of experiments designed to examine the effect on induction of pdx-1 expression of different combinations of early and late factors. Note that the results depicted in Figure 13 A represent normalized expression, and the results depicted in Figure 13B represent expression as % of actin input.
  • Figure 14 shows the effect of nodal on induction of pdx-1 expression.
  • the 2 EF - 3 LF protocol was performed in the presence or absence of 50 ng/ml of recombinant nodal protein.
  • Figure 15 shows the effect of activin and BMP4 protein concentrations on induction of pdx-1 expression. The data were determined by pdx-1 and actin standard curves, and are expressed as % actin.
  • Figure 16 shows the effect of activin and BMP4 protein concentrations on induction of expression of a number of endocrine genes. Pdx-1 and insulin gene expression were calculated based on standard curves and expressed as % actin. Pax4, somatostatin, and glucagon were calculated as relative values.
  • Figures 17A-17D show that the 2 EF -3 LF initial differentiation protocol (panels C and D) more effectively induces pdx-1 expression than the 4 EF - 4 LF initial differentiation protocol (panels A and B).
  • Figures 18A and 18B show pdx-1 expression followed by release of C- peptide from representative cultures of embryonic stem cells differentiated using an extended differentiation protocol including both an initial differentiation phase and a maturation phase.
  • Figure 18A is a schematic representation of the combined protocol.
  • the left panel of Figure 18B shows the expression of pdx-1 by quantitative PCR following the first 20 days of differentiation (the initial differentiation protocol).
  • the right panel of Figure 18B shows release of C-peptide at day 36 of differentiation. Day 36 is approximately halfway through the maturation portion of the extended differentiation protocol.
  • FIGS 19A and 19B show release of C-peptide from cultures assayed at various stages during the extended differentiation protocol.
  • Figures 20A-20F show insulin expression by in situ hybridization.
  • Figures 2OA and 2OB show that after 20 days in the initial differentiation protocol, embryoid bodies contain a few isolated insulin 4* cells.
  • Figures 2OC and 2OD show that further differentiation using the maturation protocol induces insulin expression in a higher percentage of cells in the embryoid body. Additionally, following the maturation protocol, the insulin 4* cells appear most prevalent within sectors / clusters within the embryoid body.
  • Figure 2OE shows a cryosection of a day 20 embryoid body.
  • Figure 2OF shows a sense strand negative control.
  • Figures 21A-21F show C-peptide protein expression by immunocytochemistry in day 45 embryoid bodies.
  • Figures 22A-22E show pdx-1 expression by in situ hybridization.
  • Figures 22 A and 22B show pdx-1 expression in embryoid bodies cultured in the initial differentiation protocol for 20 days.
  • Figure 22C show that embryoid bodies cultured for 20 days in the absence of growth factors fail to express pdx-1.
  • Figure 22D summarizes the results of the experiments depicted in Figures 22 A and 22C, and confirms robust pdx-1 expression in cells cultured for 20 days in the presence (left) versus the absence (right) of growth factors.
  • Figure 22E shows that after 43 days in a combination of the initial and maturation protocols, embryoid bodies robustly express pdx-1.
  • Pdx-1 expression is generally clustered to a portion of a particular embryoid body.
  • Figure 23 shows two variations of the multi-step maturation protocol that result in C-peptide release.
  • the top diagram is identical to that shown in Figure 19.
  • the middle and bottom diagrams show two variations, each more efficient than the top diagram protocol.
  • Figure 24 shows the release of C-peptide when variations of the multi-step protocol was used.
  • FIGS 25A-25C show the effect of forskolin in Step 4 of the multi-step maturation protocol on the release of C-peptide.
  • FIGS 26A and 26B show the effect of fetal bovine serum (FBS) in Step 4 of the multi-step protocol on the release of C-peptide.
  • FBS fetal bovine serum
  • Figures 27A-27D show the protocol used (Figure 27A), the effect of glucose concentration on differentiated HES3 cells measured by the release of C-peptide (Figure 27B), pdx-1 mRNA (Figure 27C) and insulin mRNA (Figure 27D).
  • Figure 28 shows the expression of Pdx-1 and C-peptide by single and double immunohistochemistry in differentiated HES3 embryoid bodies.
  • Figure 29 shows the expression of Pdx-1 on Day 20 of MATRIGELTM differentiation in the presence and absence of growth factors, various late factors and early factors.
  • Figure 30 shows the expression of Pdx-1 in the presence and absence of MATRIGELTM between Day 0 and Day 10.
  • Figure 31 shows the release of C-peptide on day 26 and day 29 when a simplified multi-step maturation protocol was used.
  • Figure 32 shows the presence of insulin and C-peptide by double immunofluorescence in sectioned embryoid bodies.
  • the top panels are high magnification images and the bottom panels are low magnification images.
  • Figure 33 shows the expression of Pdx-1 and Nkx ⁇ .l, both differentiation markers of ⁇ -cell endocrine lineage.
  • Diabetes mellitus is a common disease characterized by the inability to regulate circulating glucose levels due to problems with insulin production or utilization.
  • Type I diabetes (about 5% of all diabetes cases) is caused by the autoimmune destruction of the pancreatic beta cell that produces insulin.
  • the more common type 2 diabetes, associated with obesity, has many causes related to either a decreased insulin output by the pancreas or to inefficient utilization of insulin at the target organs (insulin resistance).
  • insulin resistance insulin resistance
  • the present invention provides a variety of methods to direct the differentiation of embryonic stem cells to a pancreatic cell fate. These include methods comprising the use of several early and late factors (EF and LF) administered over an approximately 20 day time frame.
  • This initial differentiation methodology promotes expression of pdx-1 and promotes differentiation of embryonic stem cells along a pancreatic lineage. Additionally, this initial differentiation methodology promotes expression of markers of terminal pancreatic differentiation, such as insulin and somatostatin, though at a lower level than that of pdx-1.
  • the present invention provides a variety of experiments identifying factors and optimized sub-sets of early and late factors that help promote differentiation of embryonic stem cells along a pancreatic lineage.
  • the present invention provides maturation protocols designed to further promote the differentiation of stem cells along a pancreatic lineage.
  • the invention provides maturation protocols that can be used to promote terminal differentiation of embryonic stem cells that were previously directed along a pancreatic lineage using the initial differentiation protocols detailed herein.
  • embryonic stem cells can be further differentiated to induce and/or increase expression of terminal differentiation markers including, but not limited to, insulin and C-peptide.
  • such maturation protocols can be used to produce cell or cell clusters that are glucose responsive (e.g., mimic a function of pancreatic beta cells).
  • an element means one element or more than one element.
  • protein is a polymer consisting essentially of any of the 20 amino acids.
  • polypeptide is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied.
  • polynucleotide sequence and “nucleotide sequence” are also used interchangeably herein.
  • Recombinant means that a protein is derived from a prokaryotic or eukaryotic expression system.
  • wild type refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.
  • mutant refers to any change in the genetic material of an organism, in particular a change (i.e., deletion, substitution, addition, or alteration) in a wild-type polynucleotide sequence or any change in a wild-type protein sequence.
  • variant is used interchangeably with “mutant”.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • a polynucleotide sequence (DNA, RNA) is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein.
  • tissue-specific promoter means a nucleic acid sequence that serves as a promoter, i.e., regulates expression of a selected nucleic acid sequence operably linked to the promoter, and which affects expression of the selected nucleic acid sequence in specific cells of a tissue, such as cells of neural origin, e.g. neuronal cells.
  • tissue-specific promoter also covers so-called “leaky” promoters, which regulate expression of a selected nucleic acid primarily in one tissue, but cause expression in other tissues as well.
  • Homology and “identity” are used synonymously throughout and refer to sequence similarity between two peptides or between two nucleic acid molecules.
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. A degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • a “chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence defining a domain ⁇ e.g. polypeptide portion) foreign to and not substantially homologous with any domain of the first polypeptide.
  • a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
  • small organic molecule refers to compounds smaller than proteins that are generally characterized by the ability to transit cellular membranes more easily than proteins. Preferred small organic molecules are characterized as having a size less than 10,000 AMU. More preferably, between 5000-10,000 AMU. Most preferably, the small organic molecules are characterized as having a size between 1000-5000 AMU.
  • non-human animals include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.
  • proliferating and “proliferation” refer to cells undergoing mitosis.
  • “Differentiation” in the present context means the formation of cells expressing markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells incapable of further division or differentiation.
  • the pathway along which cells progress from a less committed cell, to a cell that is increasingly committed to a particular cell type, and eventually to a terminally differentiated cell is referred to as progressive differentiation or progressive commitment.
  • Cell which are more specialized (e.g., have begun to progress along a path of progressive differentiation) but not yet terminally differentiated are referred to as partially differentiated.
  • progenitor cell is used synonymously with “stem cell”. Both terms refer to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells, hi a preferred embodiment, the term progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells maybe considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors.
  • embryonic stem cell is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see US Patent Nos. 5843780, 6200806). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, US Patent Nos. 5945577, 5994619, 6235970).
  • the distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
  • adult stem cell is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue.
  • Stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture.
  • Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As indicated above, stem cells have been found resident in virtually every tissue. Accordingly, the present invention appreciates that stem cell populations can be isolated from virtually any animal tissue.
  • tissue refers to a group or layer of similarly specialized cells which together perform certain special functions.
  • substantially pure with respect to a particular cell population, refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to the cells making up a total cell population.
  • the terms "substantially pure” or "essentially purified”, with regard to a preparation of one or more partially and/or terminally differentiated cell types refer to a population of cells that contain fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are either undifferentiated, are differentiated to a non-endodermal cell type, or are differentiated to an endodermal tissue type that is not functionally or structurally related to that of the essentially purified population of cells.
  • a "marker” is used to determine the state of a cell.
  • Markers are characteristics, whether morphological or biochemical (enzymatic), particular to a cell type, or molecules expressed by the cell type.
  • markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art.
  • a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids.
  • a marker may comprise a morphological or functional characteristic of a cell. Examples of morphological traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages.
  • Markers may be detected by any method available to one of skill in the art.
  • markers may be detected using analytical techniques, such as by protein dot blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or any other gel system that separates proteins, with subsequent visualization of the marker (such as Western blots), gel filtration, affinity column purification; morphologically, such as fluorescent- activated cell sorting (FACS), staining with dyes that have a specific reaction with a marker molecule (such as ruthenium red and extracellular matrix molecules), specific morphological characteristics (such as the presence of microvilli in epithelia, or the pseudopodia/filopodia in migrating cells, such as fibroblasts and mesenchyme); and biochemically, such as assaying for an enzymatic product or intermediate, or the overall composition of a cell,
  • FACS fluorescent- activated cell sorting
  • nucleic acid markers any known method may be used. If such a marker is a nucleic acid, PCR, RT-PCR, in situ hybridization, dot blot hybridization, Northern blots, Southern blots and the like may be used, coupled with suitable detection methods. If such a marker is a morphological and/or functional trait, suitable methods include visual inspection using, for example, the unaided eye, a stereomicroscope, a dissecting microscope, a confocal microscope, or an electron microscope. The invention contemplates methods of analyzing the progressive or terminal differentiation of a cell employing a single marker, as well as any combination of molecular and/or non-molecular markers.
  • Differentiation is a developmental process whereby cells assume a specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types.
  • the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell).
  • terminally differentiated cell In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the terminally differentiated cells lose or greatly restrict their capacity to proliferate.
  • differentiation refers to cells that are more specialized in their fate or function than at a previous point in their development, and includes both cells that are terminally differentiated and cells that, although not terminally differentiated, are more specialized than at a previous point in their development.
  • the development of a cell from an uncommitted cell (for example, a stem cell), to a cell with an increasing degree of commitment to a particular differentiated cell type, and finally to a terminally differentiated cell is known as progressive differentiation or progressive commitment. Cells which have become more specialized but are not yet terminally differentiated are referred to as partially differentiated.
  • initiation protocol or "initiation method” are used interchangeably to refer to any of the various methods of the invention used to begin biasing embryonic stem cells and embryoid bodies along a pancreatic lineage.
  • the initiation protocol is typically approximately 20 days and includes addition of early factors (EF) and late factors (LF).
  • EF early factors
  • LF late factors
  • initiation protocols of shorter durations, for example 10 days in the presence of only EFs are also contemplates
  • Exemplary initiation protocols include, but are not limited to, the eight factor protocol comprising addition of 4 EFs and 4 LFs, as well as the 2 EF-3 LF protocol.
  • initiation protocols are also referred to more specifically according to the number or combination of early and late factors used to help promote initial differentiation of embryonic stem cells along a pancreatic lineage.
  • maturation protocol is used to refer to any of the various methods used to further differentiate embryonic stem cells and embryoid bodies previous subjected to the initiation protocol.
  • the maturation protocol can be subdivided into various stages, and the term maturation protocol will be used to refer to methods where the cells are subjected to any or all of these various phases. Specific reference to the number of days in culture, the stage of the protocol, or the factors added will be used to help distinguish the various permutations and stages of the maturation protocol(s).
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.
  • systemic administration "administered systemically,”
  • peripheral administration and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • hedgehog signaling means hedgehog signaling
  • hedgehog signal transduction means hedgehog signaling pathway
  • hedgehog signaling pathway refers to the mechanism whereby hedgehog proteins (Sonic, Desert, Indian hedgehog) influence proliferation, differentiation, migration, and survival of diverse cell types (see, for example, Allendoerfer (2003) Current Opinion Investig. Drugs 3: 1742-1744; Ingham (2001) Genes & Dev 15: 3059-3087).
  • Hedgehog agonists or “agonists of hedgehog signaling.”
  • Agents that inhibit hedgehog signal transduction are referred to as “hedgehog antagonists” or “antagonists of hedgehog signaling.”
  • Hedgehog signal transduction may be influenced by hedgehog proteins, or by agents that agonize or antagonize hedgehog signaling at any point in the pathway (extracellularly, at the cell surface, or intracellularly). For further examples see U.S. Patent No. 6,444,793; U.S. Patent No. 6,683,108; U.S. Patent No. 6,683,198; U.S. Patent No.
  • BMP signaling BMP signal transduction
  • BMP signaling pathway BMP signaling pathway
  • BMP agonists agents that promote BMP signal transduction
  • BMP antagonists agents that inhibit BMP signal transduction
  • BMP signal transduction may be influenced by BMP proteins, or by agents that agonize or antagonize BMP signaling at any point in the pathway (extracellularly, at the cell surface, or intracellularly).
  • Wnt signaling Wnt signal transduction
  • Wnt signaling pathway Wnt signaling pathway
  • Wnt agonists or “agonists of Wnt signaling.”
  • Wnt antagonists or “antagonists of Wnt signaling.”
  • Wnt signal transduction may be influenced by Wnt proteins, or by agents that agonize or antagonize Wnt signal transduction at any point in the pathway (extracellularly, at the cell surface, or intracellularly).
  • Notch signaling As used interchangeably throughout the application to refer to the mechanism whereby Notch proteins influence proliferation, differentiation, migration, and survival of diverse cell types (see, for example, Baron, Stem Cell Dev. Bio. 14: 113-119, 2003).
  • Notch agonists agents that promote Notch signal transduction
  • Notch antagonists agents that inhibit Notch signal transduction
  • Notch signal transduction may be influenced by a Notch proteins, or by agents that agonize or antagonize Notch signal transduction at any point in the pathway (extracellularly, at the cell surface, or intracellularly).
  • adherent matrix refers to any matrix that promotes adherence of cells in culture (e.g., fibronectin, collagen, laminins, superfibronectin).
  • exemplary matrices include MATRIGELTM (Beckton-Dickinson), HTB9 matrix, and superfibronectin.
  • MATRIGELTM is derived from a mouse sarcoma cell line.
  • HTB9 is derived from a bladder cell carcinoma line (US Patent 5,874,306).
  • pancreas is art recognized, and refers generally to a large, elongated, racemose gland situated transversely behind the stomach, between the spleen and duodenum.
  • the pancreatic exocrine function e.g., external secretion, provides a source of digestive enzymes.
  • pancreatin refers to a substance from the pancreas containing enzymes, principally amylase, protease, and lipase, which substance is used as a digestive aid.
  • the exocrine portion is composed of several serous cells surrounding a lumen.
  • These cells synthesize and secrete digestive enzymes such as trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospholipase A2, elastase, and amylase.
  • digestive enzymes such as trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospholipase A2, elastase, and amylase.
  • the endocrine portion of the pancreas is composed of the islets of Langerhans.
  • the islets of Langerhans appear as rounded clusters of cells embedded within the exocrine pancreas.
  • the ⁇ cells constitute about 20% of the cells found in pancreatic islets and produce the hormone glucagon.
  • Glucagon acts on several tissues to make energy available in the intervals between feeding. In the liver, glucagon causes breakdown of glycogen and promotes gluconeogenesis from amino acid precursors.
  • the ⁇ cells produce somatostatin which acts in the pancreas to inhibit glucagon release and to decrease pancreatic exocrine secretion.
  • pancreatic polypeptide The hormone pancreatic polypeptide (PP) is produced in the ⁇ cells. This hormone inhibits pancreatic exocrine secretion of bicarbonate and enzymes, causes relaxation of the gallbladder, and decreases bile secretion.
  • the major target organs for insulin are the liver, muscle, and fat-organs specialized for storage of energy.
  • pancreatic duct includes the accessory pancreatic duct, dorsal pancreatic duct, main pancreatic duct and ventral pancreatic duct. Serous glands have extensions of the lumen between adjacent secretory cells, and these are called intercellular canaliculi.
  • interlobular ducts refers to intercalated ducts and striated ducts found within lobules of secretory units in the pancreas.
  • the "intercalated ducts” refers to the first duct segment draining a secretory acinus or tubule. Intercalated ducts often have carbonic anhydrase activity, such that bicarbonate ion may be added to the secretions at this level.
  • “Striated ducts” are the largest of the intralobular duct components and are capable of modifying the ionic composition of secretions.
  • islet equivalents or "IEs” is a measure used to compare total insulin content across a population or cluster of cells.
  • An islet equivalent is defined based on total insulin content and an estimate of cell number which is typically quantified as total protein content. This allows standardization of the measure of insulin content based on the total number of cells within a cell cluster, culture, sphere, or other population of cells.
  • the standard rat and human islet is approximately 150 ⁇ m in diameter and contains 40-60 ng insulin/ ⁇ g of total protein.
  • human islet- like structures differentiated by the methods of the present invention contain approximately 50 ng insulin/ ⁇ g of total protein.
  • reporter construct is used to refer to constructs that 'report' or
  • reporter constructs include portions of the promoter, enhancer, or other regulatory sequences of a particular gene sufficient to regulate expression in a developmentally relevant manner.
  • Such regulatory sequences are operably linked to a nucleic acid sequence encoding a marker that can be readily detectable (the 'reporter gene'). In this way, expression of a readily detectable product can be monitored, and this product is regulated in a manner consistent with the promoter or enhancer to which it is operably linked.
  • Reporter genes may be introduced into cells by any of a number of ways including transfection, electroporation, micro-injection, etc.
  • reporter genes include, but are not limited to, green fluorescent protein (GFP), recombinantly engineered variants of GFP, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, LacZ, luciferase, firefly Remmila protein.
  • Further exemplary reporter genes encode antibiotic resistance proteins including, but not limited to, neomycin, hygromycin, zeocine, and puromycin.
  • xeno-free / clinically compliant ES cells or cell lines or "xeno- free CGMP-compliant hES lines” refers to
  • hESC human embryonic stem cell lines approved for U.S. government federal research funding have been derived and propagated on mouse embryonic fibroblasts (MEFs) and in the presence of culture medium containing animal-based ingredients.
  • MEFs mouse embryonic fibroblasts
  • CGMP current good manufacturing practice
  • GTCP good tissue culture practice
  • embryonic stem cells from any of a variety of species are well known in the art. Exemplary species include, but are not limited to, mice, non-human primates, and humans. Furthermore, under a variety of circumstances, many have observed the differentiation of embryonic stem cells to any of a number of partially or terminally differentiated cell types. For example, embryonic stem cells aggregated to form embryoid bodies may produce embryoid bodies that include small regions or foci of beating tissue. This beating tissue indicates that a small percentage of cells in the embryoid body have differentiated to form cardiomyocytes.
  • the challenge is not to wait patiently as embryonic stem cells randomly differentiate along particular lineages.
  • the challenge to devise methods of differentiating embryonic stem cells that produce a disparate "mixed bag" of cell types across a culture.
  • the challenge is to develop efficient methods to direct the differentiation of embryonic stem cells to particular cell types or along particular developmental lineages. Such methods are essential to increase our understanding of stem cell biology, to produce substantially purified cultures of differentiated cell types, and to develop therapeutics based upon differentiated cells.
  • the present invention addresses the limitations in the prior art and offers methodologies for directing the differentiation of embryonic stem cells to endodermal cell types.
  • the methods of the present invention can be used to direct the differentiation of embryonic stem cells to produce various partially and/or terminally differentiated cells or cell clusters.
  • the methods of the present invention e.g., the initiation protocols, the maturation protocols, and combinations thereof
  • the methods of the present invention can be used to direct the differentiation of embryonic stem cells to partially and terminally differentiated pancreatic cell types.
  • Partially and terminally differentiated cell types for example pancreatic cell types, induced by the initiation and/or maturation protocols of the present invention can be further expanded and/or purified to produce essentially purified cultures of one or more partially and/or terminally differentiated endodermal cell types.
  • the methods of the present invention can be used to produce, from embryonic stem cells, (i) essentially purified populations of terminally differentiated pancreatic cell types (e.g., either a single terminally differentiated pancreatic cell type or multiple terminally differentiated pancreatic cell types); (ii) essentially purified populations of partially differentiated pancreatic cell types (e.g., either a single partially differentiated pancreatic cell type or multiple partially differentiated pancreatic cell types); or (iii) essentially purified populations of one or more partially and/or terminally differentiated pancreatic cell types.
  • terminally differentiated pancreatic cell types e.g., either a single terminally differentiated pancreatic cell type or multiple terminally differentiated pancreatic cell types
  • partially differentiated pancreatic cell types e.g., either a single partially differentiated pancreatic cell type or multiple partially differentiated pancreatic cell types
  • essentially purified populations of one or more partially and/or terminally differentiated pancreatic cell types essentially purified populations of one or
  • Cultures of embryonic stem cells can be differentiated using methods that include a step involving formation of embryoid bodies or directly (e.g., without a step involving the formation of embryoid bodies).
  • an early step in the differentiation process comprises the aggregation of embryonic stem cells to form embryoid bodies.
  • Embryonic stem (ES) cells can be differentiated by removing the cells from the feeder layer and aggregating them in suspension to form embryoid bodies (EBs).
  • EBs can be made by plating dissociated ES cells in bulk on low-attachment plates or by the hanging drop method.
  • ES cells may be dissociated fully into single cells or partially into small clumps by a number of methods including trypsin, collagenase, dispase, EDTA, or mechanical disruption.
  • the method of dissociation can be readily selected by one of skill in the art and may vary depending on the species from which the cells are derived, as well as the overall health of the cells. For example, human ES cells do not survive as well following dissociation to the single cell level.
  • the dissociation technique can be selected so as to remove the ES cells from the feeder layer without dissociating the cells to the single cell level prior to EB formation.
  • the EBs can be cultured in suspension (e.g., as floating aggregates of cells, on filters, or embedded in gel-like matrices) for a period of time, preferably ranging from 3 days to 3 weeks. In certain embodiments, EBs can be cultured in suspension for less than 3 days, for example, for 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours. In certain other embodiments, the EBs can be cultured for greater than 3 weeks.
  • ES cells from certain species appear to be more sensitive to the level of dissociation achieved prior to EB formation. Accordingly, in addition to the above outlined approach in which certain ES cells are dissociated less completely (e.g., not dissociated to single cells) prior to EB formation, the present invention contemplates methods of EB formation in which ES cells are dissociated in the presence of agents that block apoptosis or otherwise promote cell survival. Exemplary agents include, but are not limited to, caspase inhibitors.
  • EBs can be cultured in a variety of media including, but not limited to, basal media BME, CMRLl 066, MEM, DMEM, DMEM/F12, RPMI, Glasgow MEM with or without alpha modification, IMDM, Leibovitz's L-15, McCoys 5A, Media 199, Ham's F-10, Ham's F-12, F-12K, NCTC- 109 medium, Waymouth's media, William's Media E, or a combination of any of the above.
  • any of the foregoing media can be supplemented with varying concentration of glucose (1-50 mM), sodium pyruvate, non-essential amino acids, nucleosides, N-2 supplement, G-5 supplement, and B27 supplement.
  • the media may or may not contain phenol-red.
  • the media can be buffered with an appropriate amount of buffering salt.
  • Exemplary buffering salts include, but are not limited to, sodium bicarbonate, Tris, HEPES, sodium acetate.
  • the pH of the EB media can vary between 5 and 9.
  • the culture media may be supplemented with different amounts of animal serum.
  • Exemplary animal sera commonly used in the art include, but are not limited to, fetal bovine serum (FBS), bovine serum (BS), horse serum (HS), chicken serum (CS), goat serum (GS).
  • the serum may or may not be heat-activated.
  • the media may be supplemented with a chemically defined serum replacement, such as Knockout DMEM with Knockout Serum Replacement.
  • the concentration of animal serum or serum replacement in the media is selected in the range from 0% to 20%. hi other embodiments, the concentration of serum or serum replacement in the media is greater than 20%, for example, is between 20% - 40%.
  • the EBs can be cultured in basal media supplemented with serum or serum replacement alone, EBs may also be cultured in media further containing media conditioned by another cell line. Alternatively, in another embodiment, EBs can be cultured in basal media lacking serum or serum replacement, but containing media conditioned by another cell line.
  • Exemplary cell lines from which conditioned media can be obtained include, but are not limited to, mouse embryonic fibroblasts (MEFs); mouse or human insulinomas (e.g., RIN-5, beta-TC, NIT-I, INS-I, INS-2); hepatomas (e.g., HepG2, Huh-7, HepG3); HT-1080; endothelial cells (e.g., HUVEC); bone marrow stromal cells; visceral endoderm-like cells such as end-2; or mesenchymal cells such as HEPM or 7F2.
  • mouse embryonic fibroblasts MEFs
  • mouse or human insulinomas e.g., RIN-5, beta-TC, NIT-I, INS-I, INS-2
  • hepatomas e.g., HepG2, Huh-7, HepG3
  • HT-1080 e.g., endothelial cells
  • endothelial cells e.g
  • conditioned media can be obtained from cultured embryonic, fetal, or adult tissues (e.g., derived from human, non-human primate, mouse, or other animals), or from a primary cell line established from a particular tissue type (e.g., pancreas, liver, bone marrow, lung, skin, blood, etc.) and derived from an animal (e.g., human, non- human primate, mouse, or other animal).
  • tissue type e.g., pancreas, liver, bone marrow, lung, skin, blood, etc.
  • an animal e.g., human, non- human primate, mouse, or other animal.
  • Embryonic tissues include endoderm, mesoderm, ectoderm, and/or extraembryonic tissue such as trophectoderm and visceral endoderm.
  • the embryonic tissue is chosen based on the ability of that tissue to send instructive signals to the embryonic pancreas during development.
  • Such instructive tissues include notochord and dorsal aorta.
  • Exemplary primary cell lines include endothelial cells, aortic smooth muscle cells, mesenchymal cells, endocrine or exocrine cells of the pancreas, hepatocytes, intestinal epithelial cells, and ductal cells.
  • Media can also be conditioned from any cells derived from mouse embryonic stem cells.
  • the conditioned media may be derived from a cell line, tissue, etc. of the same species as the EBs or from a different species.
  • the foregoing media constitutes the starting point for directing the differentiation of the cells to a particular differentiated endodermal cell type.
  • Any of the foregoing media can now be further supplemented with appropriate differentiation factors to direct cells in the EBs to differentiate along particular endodermal lineages such as the pancreatic lineage, hepatic lineage, lung lineage, etc.
  • EBs can be cultured in media supplemented with particular differentiation factors to direct the differentiation of cells in the EBs to pancreatic cell types including insulin-producing cells.
  • Such factors include but are not limited to activin A, activin B, BMP2, BMP4, nodal, TGF ⁇ , sonic hedgehog, desert hedgehog, EGF, HGF, FGF2, FGF4, FGF8, FGFl 8, PDGF, Wnt proteins, retinoic acid, sodium butyrate, NGF, HGF, GDF, growth hormone, PYY, cardiotropin, GLP- 1, exendin-4, betacellulin, nicotinamide, tri-iodothyroxine, insulin, IGF-I, IGF-II, placental lactogen, VEGF, wortmannin, gastrin, cholecystokinin, Sphingosine-1- phosphate, FGF-IO, FGF inhibitors, growth hormone, KGF, islet neogenesis- associated protein (INGAP), Reg, and factors that increase cAMP levels such as forskolin and IBMX.
  • activin A activin B, BMP2, BMP4, nodal,
  • the invention contemplates that, for certain of the above referenced protein factors, small molecule agonists that mimic the bioactivity of the protein are known in the art. Such small molecule mimics may function in any of a number of ways to produce similar biological consequences as the protein. Accordingly, the invention contemplates methods in which the EBs are cultured in the presence of small molecule agonists/mimics of any of the foregoing proteins.
  • certain of these factors may influence cell fate by binding to receptors on the surface of the cells, and thereby modulating one or more signal transduction pathways functional in the cells.
  • certain of these factors may influence cell fate by transiting the cell membrane and acting intracellularly to modulate one or more signal transduction pathways functional in the cells.
  • the invention contemplates using one or more of these factors to help promote the differentiation of cells to pancreatic cell types.
  • the one or more factors influence the cells by modulating the same signal transduction pathway (e.g., Sonic hedgehog protein in combination with Desert hedgehog protein), hi another embodiment, the one or more factors influence the cells by modulating different signal transduction pathways (e.g., one or more hedgehog proteins in combination with one or more Wnt proteins), hi another embodiment, one or more factors influence cells via mechanisms that may or may not overlap.
  • the invention contemplates that one or more of the above differentiation factors can be added to a culture of EBs to help promote their differentiation to pancreatic cell types. When more than one differentiation factor is added to the culture, the invention contemplates that the differentiation factors can be added concomitantly or concurrently.
  • the experiments summarized herein provide multiple examples of initiation protocols that bias embryonic stem cells along a pancreatic lineage. Furthermore, the experiments summarized herein provide multiple examples of maturation protocols that, when used in combination with an initiation protocol, help promote the further differentiation of biased embryonic stem cells to terminally differentiated pancreatic cell type (e.g., produce cells that express one or more markers indicative of a terminally differentiated pancreatic cell type).
  • the EBs can be replated on an adherent matrix.
  • adherent matrices include, but are not limited to, gelatin, MATRIGELTM, various types of collagens, laminins, fibronectins, or a combination of any of the foregoing.
  • EBs can be replated directly onto the adherent matrix or EBs can be dissociated prior to replating onto the adherent matrix. Directed Differentiation
  • ES cells can be differentiated without a step including EB formation.
  • ES cells can be plated directly on an appropriate adherent matrix without first forming cultures of EBs.
  • the ES cells can also be differentiated either as a monolayer in culture or on feeder cells.
  • the ES cells can be plated in any of the above referenced combination of media appropriate for the culture of EBs and further supplemented with one or more of the differentiation factors outlined above.
  • Exemplary adherent matrices include, but are not limited to, gelatin, MATRIGELTM, various types of collagens, laminins, fibronectins, or a combination of any of the foregoing.
  • the invention contemplates that the ES cells, differentiating ES cells, or EBs can be cultured either under standard tissue culture conditions of oxygen and carbon dioxide, or in an incubator where oxygen tension can be varied.
  • EBs can be cultured either in suspension in liquid media or in suspension by embedding in a 2D or 3D gel or matrix.
  • exemplary matrices include, but are not limited to, MATRIGELTM, collagen gel, laminin gel, as well as artificial 3D lattices constructed from materials such as polylactic acid or polyglycolic acid.
  • differentiation factors can be administered either by addition to the surrounding liquid medium or by covalently or non-covalently linking the factors to the particular matrix in which the EBs are suspended.
  • EBs can be cultured on a Transwell. Culture on a Transwell may facilitate establishment of cell polarity.
  • differentiation of ES cells or EBs can be promoted by co-culturing the cells with cells, cell lines, or tissues of the endoderm or with cells, cell lines, or tissues derived from tissues known to induce endodermal differentiation during development.
  • the invention contemplates that a single differentiation step likely will not produce the particular partially or terminally differentiated cell type desired, or will not necessarily produce them in the desired ratios or percentages. Accordingly, the invention contemplates that ES cells and EBs can be cultured and differentiated in stages. At each successive stage, the differentiation factors and differentiation matrices may be the same or different.
  • Progressive differentiation of ES cells and EBs can be measured by examining markers of partially and/or terminally differentiated cells of the particular tissue of interest. For example, in methods where the goal is the differentiation of ES cells (with or without the formation of EBs) to pancreatic cell type, progressive differentiation can be monitored by assaying expression of markers of partially or terminally differentiated pancreatic cells (e.g., early markers of the endodermal lineage; early markers of the pancreatic lineage; markers of partially differentiated endocrine pancreatic cells; markers of partially differentiated exocrine pancreatic cells; markers of terminally differentiated endocrine pancreatic cells; markers of terminally differentiated exocrine pancreatic cells).
  • markers of partially or terminally differentiated pancreatic cells e.g., early markers of the endodermal lineage; early markers of the pancreatic lineage; markers of partially differentiated endocrine pancreatic cells; markers of partially differentiated exocrine pancreatic cells; markers of terminally differentiated endocrine pancre
  • early differentiation to definitive endoderm could be monitored by assaying expression of genes including, but not limited to, soxl7, HNFl ⁇ , HNF3 ⁇ , HNF3 ⁇ , HNF3 ⁇ , brachyury T, goosecoid, claudins, AFP, HEX, eomesodermin, TCF2, Mixll, CXCR4, GAT A5, and proxl.
  • differentiation into non-endodermal lineages could be monitored by assaying expression of non-endodermal genes, for example, genes indicative of extraembryonic tissue.
  • pancreatic genes e.g., exocrine pancreatic genes and endocrine pancreatic genes
  • pancreatic genes including, but not limited to,pdx-l, ngn3, HB9, HNF6, ptfl-p48, islet 1, nkx ⁇ .l, nkx2.2, glut2, neuroD, cytokeratin 19, LAPP, pax4, pax6, HESl, amylase, glucagon, somatostatin, insulin, hormone convertase, glucokinase, Sur-1, Kirb6.2 and pancreatic polypeptide.
  • gene expression can be measured in living cells over time to provide a snapshot of differentiation in a given culture.
  • samples of cells from a given culture at a given time can be taken and processed. Such cells would provide a representation of the differentiation in a particular culture at a particular time.
  • Gene expression can be measured by a variety of techniques well known in the cell biological and molecular biological arts. These techniques include RT-PCR, northern blot analysis, in situ hybridization, microarray analysis, SAGE, or MPSS. Protein expression can similarly be analyzed using well known techniques such as western blot analysis, immunohistochemical staining, ELISA, or RIA.
  • Differentiation into definitive endoderm could be accomplished by activating certain pathways including but not limited to nodal, wnt, and FGF signaling.
  • Nodal belongs to the TGF-beta superfamily of ligands that include activins and BMPs. Addition of these TGF ⁇ related ligands could drive hES cells towards definitive endoderm.
  • Wnt signaling could be activated by addition of any of the Wnt ligands.
  • wnt signaling is the stabilization of ⁇ -catenin. Stabilization of ⁇ -catenin could also be accomplished by addition of GSK3 inhibitors, including but not limited to derivatives of 6-bromoindirubin.
  • Inhibition of FGF signaling may also help to direct ES cells down the endodermal pathway. Inhibition of FGF signaling could be accomplished by using one of several FGF receptor antagonists such as the compound SU5402. Induction of endodermal differentiation can be assessed by measuring the phosphorylation state of intracellular Smad2 protein.
  • Differentiation towards endocrine pancreas could be biased by expression of key developmental genes in ES cells.
  • expression of pdx-1 under an appropriate promoter could be used to drive ES down the pancreatic lineage and help bias the cells to respond to the differentiation factors.
  • the promoter could be a constitutive one such as CMV, SV40, EFl ⁇ , or beta-actin.
  • the promoter could be an inducible one such as metallothionin, ecdysone, or tetracycline.
  • the recombinant protein could also be tagged to a regulatory element such as the ligand-binding domain of the estrogen receptor or variants thereof.
  • Such a fusion protein could be regulated by addition or withdrawal of estrogen analogs including tomaxifin.
  • Recombinant DNA could be introduced in ES cells using a variety of methods including electroporation, lipofection, or transduction by viral agents such as adenovirus, lentivirus, herpes virus, or other pleiotropic viruses.
  • inhibition of certain genes may help promote differentiation and/or help promote responsiveness of the ES to the differentiation factors.
  • inhibition of the smoothed/patched receptor, RBK-JK, or HESl in hES-derived cells could help drive the ES cells toward the pancreatic lineage.
  • Inhibition of the genes could be accomplished by antisense oligos, siRNA, deletion of endogenous alleles by homologous recombination or constitutive expression of an inhibitor or dominant negative gene.
  • essentially purified preparations of one or more partially or terminally differentiated cell types of a particular tissue can be generated directly from ES cells or EBs.
  • selection can be used to further purify a preparation of cells or can be used to, for example, take a preparation that includes multiple partially and/or terminally differentiated cell types and prepare a preparation that contains fewer, or even a single, partially and/or terminally differentiated cell types.
  • Cells differentiated from ES cells may need to be expanded and selected.
  • a drug resistant marker such as neo R under the control of a particular tissue specific promoter (e.g., the insulin promoter or pdx- 1 promoter) in ES cells.
  • the selection drug like G418 can be added to select for cells expressing the marker gene.
  • G418 can be added to select for cells expressing the marker gene.
  • a suicide gene such as diphtheria toxin to a particular promoter so as to eliminate cells that differentiate along an undesired lineage.
  • Reporter ES lines can also be used to monitor progression of differentiation.
  • a construct comprising GFP downstream of a particular promoter (e.g., the pdx-1 promoter or insulin promoter) could be introduced into ES cells.
  • Cells that express the reporter can be readily detected.
  • Other reporters genes that can be used include luciferase, alkaline phosphatase, lacZ, or CAT.
  • Useful reporter lines could comprise multiple reporters to help identify cells that have differentiated along particular lineages. Cells expressing these reporters could be easily purified by
  • the purified reporter-expressing cells can be used for genomic analysis by techniques such as microarray hybridization, SAGE, MPSS, or proteomic analysis to identify more markers that characterize the purified population. These methods can identify cells that have not differentiated along the desired lineages, as well as populations of cells that have differentiated along the desired lineages, m cultures containing too many cells that have not differentiated along the desired lineages, the desired cells may be isolated and subcultured to generate an essentially purified populations of one or more partially or terminally differentiated cell types of the desired tissue. Reporter lines could be used in a high-throughput screening assay to rapidly screen for small molecules, growth factors, matrices, or different growth conditions that could favor differentiation along particular lineages.
  • Screening platform could be 24, 48, 96, or even 384-wells. Detection method would depend on the type of reporter gene being expressed. For example, a luminescence plate reader could detect luciferase reporter and a fluorescent plate reader could detect GFP or even lacZ reporters. In addition, high content screening could be performed where automated microscopes would scan each well and measure several parameters including but not limited to the number of cells expressing the reporter gene, cell size, cell shape, and cell movement. Pdx-1 positive cells or progenitors thereof could be further differentiated into endocrine cells based on methods detailed in US patent NO. 6,610,535 or as described in patent application PCT/US03/23852, the disclosures of each of which are hereby incorporated by reference in their entirety.
  • Differentiating factors used in this protocol include but are not limited to FGFl 8, cardiotropin, PYY, forskolin, HGF, heparin, insulin, dexamethasone, follistatin, betacellulin, growth hormone, placental lactogen, EGF, KGF, IGF-I, IGF-II, VEGF, exendin-4, leptin, and nicotinamide, and notch antagonists.
  • ES cells could also be differentiated in vivo by injecting them into a SCID animal. It is well known that hES cells when injected into SCID mice could form teratomas that comprise tissues from all three germ layers. Injection into different tissues or organs may drive them down a particular lineage.
  • the SCID mouse may also have a regenerating pancreas, where it has undergone partial pancreatectomy or treatment with streptozotocin.
  • hES cells could be engrafted into different parts of an embryonic or fetal animal at various stage of development.
  • ES cells are differentiated to produce essentially purified preparation of pancreatic cells.
  • Such essentially purified preparations of pancreatic cells comprise one or more partially and/or terminally differentiated pancreatic cell type.
  • the one or more partially and/or terminally differentiated pancreatic cell types include an insulin producing cell.
  • ES-derived insulin-producing cells will preferably have the following characteristics: (i) express insulin mKNA as detected by RT-PCR, northern blot, or in situ hybridization; (ii) express insulin and C-peptide as detected by western blot or immunhistochemical staining; (iii) secrete insulin and C-peptide as detectable by ELISA or RIA; (iv) show glucose responsive insulin secretion; (v) rescue a diabetic animal (e.g., STZ- treated NOD/SCID mouse) when implanted in a suitable site of the animal.
  • a diabetic animal e.g., STZ- treated NOD/SCID mouse
  • the progenitors may be purified by selecting cells expressing one or more pancreatic development genes, as outlined in detail above.
  • the markers used for selection are preferably expressed on the cell surface so they are amenable to antibody affinity capture and sorting by either flow cytometry or magnetic cell separation.
  • markers may include dynein, gap junction membrane channel protein, integral membrane protein 2 A, CXCR4, Sur-1, Glut-2, Kir6.2, microfibrillar-associated protein 2, procollagens, tachykinin2, Thy- 1.2, tenascin C, vaninl, inward rectifier K+ channel J8, adaptor protein complex AP-I, microtubule-associated protein IB, annexin Al, CD36, CD84, clusterin, catenin delta 2, endomucin, granulin, keratin Hb5, integrin ⁇ 7, lysosomal membrane glycoprotein 2, KSPG, galectin-6, lipocortin I, mannose-binding lectin, lymphocyte antigen 64, synaptotagmin 4, thrombospondin, thrombomodulin, visinin-like 1, Fabpl, Fabp2, Slc25a5, Slc2a2, Slc7a8, Ep-cam, N-cadherin, E
  • the methods of the present invention can be used to differentiate (partially or terminally) ES cells to one or more cell types of a tissue derived from the endodermal lineage.
  • the methods of the present invention can be used to differentiate ES cells to produce essentially purified preparations of one or more partially and/or terminally differentiated cells of the pancreas or liver.
  • Such essentially purified preparations of one or more partially and/or terminally differentiated cells can be formulated in a pharmaceutically acceptable carrier and administrated to patients suffering from a condition characterized by a decrease in functional performance of a particular endodermally derived organ.
  • ES cells are differentiated to produce essentially purified preparation of pancreatic cells.
  • Such essentially purified preparations of pancreatic cells comprise one or more partially and/or terminally differentiated pancreatic cell type, m certain embodiments, the one or more partially and/or terminally differentiated pancreatic cell types include an insulin producing cell.
  • ES-derived insulin-producing cells will preferably have the following characteristics: (i) express insulin rnRNA as detected by RT-PCR, northern blot, or in situ hybridization; (ii) express insulin and C-peptide as detected by western blot or immunhistochemical staining; (iii) secrete insulin and C-peptide as detectable by ELISA or RIA; (iv) show glucose responsive insulin secretion; (v) rescue a diabetic animal (e.g., STZ- treated NOD/SCDD mouse) when implanted in a suitable site of the animal.
  • a diabetic animal e.g., STZ- treated NOD/SCDD mouse
  • the present invention provides methods for directing the differentiation of embryonic stem cells to endodermal cell types.
  • the methods provided in the present application are not limited to modulating the differentiation of embryonic stem cells.
  • Embryonic stem cell have a limitless differentiation potential.
  • this limitless potential has proved challenging to researchers trying to direct the differentiation of these cells along particular lineages, in a controlled manner, and as a commercially and therapeutically useful percentage of a cell culture.
  • many adult stem cell populations have actually proven more amenable to directed differentiation.
  • the invention contemplates that these methods will similarly be able to direct the differentiation of other adult stem cells, for example, stem cells derived from a fetal or adult animal tissue.
  • stem cells include, but are not limited to, hematopoietic stem cells, neuronal stem cells, neural crest stem cells, mesenchymal stem cells, myocardial stem cells, pancreatic stem cells, hepatic stem cells, and endothelial stem cells.
  • adult stem cells can be derived from virtually any organ or tissue including, but not limited to, tongue, skin, esophagus, brain, spinal cord, endothelium, hair follicle, stomach, small intestine, large intestine, ovary, testes, blood, bone, bone marrow, umbilical cord, lung, gall bladder, and the like, hi one embodiment, the adult stem cell is a stem cell population which can differentiate along an endodermal lineage using either the methods of the present invention or other methodologies known in the art.
  • the present invention provides a variety of methods for promoting the directed differentiation of embryonic stem cells to particular differentiated cell types.
  • the invention provides methods for promoting the directed differentiation of embryonic stem cells along a pancreatic lineage.
  • Exemplary methods result in production of cells and cell clusters expressing pdx-l+, insulin, and/or C-peptide. Furthermore exemplary methods result in production of cells and cell clusters that release C-peptide.
  • the present invention further provides substantially purified cultures of cells and cell clusters (e.g., partially or terminally differentiated cells) differentiated from embryonic stem cells. By substantially purified is meant that a culture of differentiated cells or cell clusters contains less than 20%, preferably less than 15%, 10%, 7%, 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are either undifferentiated or differentiated to a cell type of a different (e.g., a non-pancreatic lineage).
  • Substantially purified cells and cell clusters differentiated along a pancreatic lineage by the methods of the present invention can be used therapeutically for treatment of various disorders associated with injury, disease, or other decrease in the functional performance of the pancreas.
  • Substantially purified cultures of cells for use in the therapeutic methods of the invention include essentially homogenous cultures of cells (e.g., essentially all of the cells are of a particular partially or terminally differentiated cell type) or heterogenous cultures of cells.
  • the culture comprises various partially differentiated and/or terminally differentiated pancreatic cell types such as a mixture of pdx-l+ and insulin+ cell types).
  • pancreatic cell types can be used in the treatment or prophylaxis of pancreatic disorders, both exocrine and endocrine, as well as pancreatic injuries.
  • the methods of the invention can be used to produce pancreatic beta-like cells or cell clusters useful for the treatment of diabetes or other conditions of impaired glucose regulation.
  • pancreatic beta-like cells or cell clusters is meant that the cells or cell clusters express pancreatic genes including, but not limited to, pdx-1, insulin, and/or C- peptide.
  • the pancreatic beta-like cells or cell clusters secrete C-peptide and are glucose responsive.
  • the methods of the present invention can be used to generate partially or terminally differentiated cell types for the treatment of an injury to the particular organ or tissue.
  • Such injuries include, but are not limited to, blunt trauma, surgical resection, or tissue damage caused by cancer or other proliferative disorder.
  • the invention also contemplates that preparations of partially and/or terminally differentiated cell types of an endodermally derived tissue may be useful for other purposes in addition to therapeutic purposes.
  • Such cells may be useful in screens to identify non-cell agents that promote proliferation, differentiation, survival, or migration of the differentiated cells.
  • the present invention provides partially and/or terminally differentiated endodermal cell types that can be used to treat or prophylactically treat injury, disease, or other decrease in functional performance of an endodermally derived tissue.
  • Such cells can be administered directly to the affected tissue (e.g., via transplantation or injection directly or adjacent to the affected tissue).
  • Such cells can also be administered systemically (e.g., via intravenous injection) and allowed to home to the site of disease or damage (e.g., to home to the affected tissue following systemic administration).
  • preparations of partially and/or terminally differentiated cells can be administered alone or can be administered in combination with other therapies.
  • the cells can be administered concurrently to or concomitantly with one or more agents that promotes one or more of proliferation, differentiation, migration, or survival.
  • agents may, for example, help transplanted cells home to the site of damage.
  • agents may help promote the survival of both the endogenous tissue and the transplanted cells.
  • Such agents can be used to treat conditions associated, in whole or in part, by loss of, injury to, or decrease in functional performance of endodermal cell types. The following are illustrative of disease states that can be treated using preparations of cells differentiated along a pancreatic lineage from ES cells.
  • Such diseases can be treated using (i) preparations of differentiated cells alone, (ii) preparations of differentiated cells in combination with one or more non-cell based compounds or agents, or (iii) preparation of differentiated cells in combination with one or more treatment regimens appropriate for the particular disease or injury being treated.
  • Diabetes mellitus is the name given to a group of conditions affecting about
  • Insulin is a hormone produced by the beta cells in the pancreas. It regulates the transportation of glucose into most of the body's cells, and works with glucagon, another pancreatic hormone, to maintain blood glucose levels within a narrow range. Most tissues in the body rely on glucose for energy production. Diabetes disrupts the normal balance between insulin and glucose. Usually after a meal, carbohydrates are broken down into glucose and other simple sugars. This causes blood glucose levels to rise and stimulates the pancreas to release insulin into the bloodstream. Insulin allows glucose into the cells and directs excess glucose into storage, either as glycogen in the liver or as triglycerides in adipose (fat) cells.
  • glucose levels remain high in the bloodstream. This can cause both acute and chronic problems depending on the severity of the insulin deficiency. Acutely, it can upset the body's electrolyte balance, cause dehydration as glucose is flushed out of the body with excess urination and, if unchecked, eventually lead to renal failure, loss of consciousness, and death. Over time, chronically high glucose levels can damage blood vessels, nerves, and organs throughout the body. This can lead to other serious conditions including hypertension, cardiovascular disease, circulatory problems, and neuropathy. 2. Pancreatitis
  • Pancreatitis can be an acute or chronic inflammation of the pancreas. Acute attacks often are characterized by severe abdominal pain that radiates from the upper stomach through to the back and can cause effects ranging from mild pancreas swelling to life-threatening organ failure. Chronic pancreatitis is a progressive condition that may involve a series of acute attacks, causing intermittent or constant pain as it permanently damages the pancreas.
  • pancreatic digestive enzymes are created and carried into the duodenum (first part of the small intestine) in an inactive form. It is thought that during pancreatitis attacks, these enzymes are prevented or inhibited from reaching the duodenum, become activated while still in the pancreas, and begin to autodigest and destroy the pancreas. While the exact mechanisms of pancreatitis are not well understood, it is more frequent in men than in women and is known to be linked to and aggravated by alcoholism and gall bladder disease (gallstones that block the bile duct where it runs through the head of the pancreas and meets the pancreatic duct, just as it joins the duodenum).
  • pancreas tissue becomes increasingly scarred and the cells that produce digestive enzymes are destroyed, causing pancreatic insufficiency (inability to produce enzymes and digest fats and proteins), weight loss, malnutrition, ascities, pancreatic pseudocysts (fluid pools and destroyed tissue that can become infected), and fatty stools.
  • pancreatic insufficiency inability to produce enzymes and digest fats and proteins
  • weight loss malnutrition
  • ascities pancreatic pseudocysts (fluid pools and destroyed tissue that can become infected)
  • pancreatic pseudocysts fluid pools and destroyed tissue that can become infected
  • fatty stools As the cells that produce insulin and glucagons are destroyed, the patient may become permanently diabetic.
  • Pancreatic insufficiency is the inability of the pancreas to produce and/or transport enough digestive enzymes to break down food in the intestine and allow its absorption. It typically occurs as a result of chronic pancreatic damage caused by any of a number of conditions. It is most frequently associated with cystic fibrosis in children and with chronic pancreatitis in adults; it is less frequently but sometimes associated with pancreatic cancer. Pancreatic insufficiency usually presents with symptoms of malabsorption, malnutrition, vitamin deficiencies, and weight loss (or inability to gain weight in children) and is often associated with steatorrhea (loose, fatty, foul-smelling stools). Diabetes also may be present in adults with pancreatic insufficiency.
  • the dosage (e.g., what constitutes a therapeutically effective amount of differentiated cells) is expected to vary from patient to patient depending on a variety of factors.
  • the selected dosage level will depend upon a variety of factors including the specific condition to be treated, other drugs, compounds and/or materials used in combination with the particular cell-based therapy, the severity of the patient's illness, the age, sex, weight, general health and prior medical history of the patient, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the cells of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of cells of the invention will be that amount of the cells which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon factors including the patient's age, sex, and the severity of their injury or disease.
  • the pharmaceutical composition comprises cells differentiated by the methods of the present invention and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition may be administered in any of a number of ways including, but not limited to, systemically, intraperitonially, directly transplanted, and furthermore may be administered in association with hollow fibers, tubular membranes, shunts, or other biocompatible devices or scaffolds.
  • treatment is intended to encompass also prophylaxis, therapy and cure, and the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; as well as poultry and pets in general.
  • the present invention provides methods for directing the differentiation of embryonic stem cells to produce cultures of endodermally derived cells. Such cultures can optionally be further purified to enrich for particular cell types, thereby providing an essentially purified preparation of endodermally derived cell types.
  • Essentially purified preparations of partially and/or terminally differentiated endodermally derived cells can be used therapeutically to treat or prophylactically treat injuries or disease of the particular organ or tissue.
  • essentially purified preparations of pancreatic cells can be formulated in a pharmaceutically acceptable carrier and delivered to patients suffering from a condition characterized by loss in functional performance of the pancreas (e.g., diabetes).
  • the invention contemplates that the therapeutic treatment additionally comprises administering other therapeutic agents.
  • agents that inhibit cell death, promote cell survival, or promote cell migration can be administered concurrently or concomitantly with the preparation of differentiated cells.
  • the invention contemplates therapeutic methods comprising administration of the subj ect cells concurrently or concomitantly with other therapeutic regimens appropriate to treat the particular condition being treated (e.g., cells + insulin for the treatment of diabetes).
  • the invention contemplates that the cells and agents can be administrated via the same method of administration or via different methods of administration.
  • the invention contemplates that in certain embodiments, preparations of terminally and/or partially differentiated pancreatic cells will be surgically or laproscopically transplanted directly to the abdominal cavity or directly to endogenous pancreatic tissue.
  • one or more additional agents are also part of the particular treatment protocol, such agents may be similarly delivered, or may be delivered in another manner (e.g., injected intravenously, transdermally, orally, subcutaneous, etc.).
  • compositions/preparations of the present invention are formulated according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA).
  • Pharmaceutical formulations of the invention can contain the active polypeptide and/or agent, or a pharmaceutically acceptable salt thereof.
  • These compositions can include, in addition to an active polypeptide and/or agent, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active agent.
  • Preferable pharmaceutical compositions are non-pyrogenic.
  • the carrier may take a wide variety of forms depending on the route of administration, e.g., intravenous, intravascular, oral, intrathecal, epineural or parenteral, transdermal, etc. Furthermore, the carrier may take a wide variety of forms depending on whether the pharmaceutical composition is administered systemically or administered locally, as for example, via surgical transplantation, laproscopic transplantation, or via a biocompatible device ⁇ e.g., catheter, stent, wire, or other intraluminal device).
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the pharmaceutical composition is formulated for sustained-release.
  • An exemplary sustained-release composition has a semi permeable matrix of a solid biocompatible polymer to which the composition is attached or in which the composition is encapsulated.
  • suitable polymers include a polyester, a hydrogel, a polylactide, a copolymer of L-glutamic acid and ethyl-L-glutamase, non-degradable ethylene-vinyl acetate, a degradable lactic acid- glycolic acid copolymer, and poly-D+- hydroxybutyric acid.
  • Polymer matrices can be produced in any desired form, such as a film, or microcapsules.
  • sustained-release compositions include liposomally entrapped modified compositions.
  • Liposomes suitable for this purpose can be composed of various types of lipids, phospholipids, and/or surfactants. These components are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • compositions according to the invention include implants, i.e., compositions or device that are delivered directly to a site within the body and are, preferably, maintained at that site to provide localized delivery.
  • biocompatible devices for use in the various methods of delivery contemplated herein can be composed of any of a number of materials.
  • the biocompatible devices include wires, stents, catheters, balloon catheters, and other intraluminal devices. Such devices can be of varying sizes and shapes depending on the intended vessel, duration of implantation, particular condition to be treated, and overall health of the patient. A skilled physician or surgeon can readily select from among available devices based on the particular application.
  • exemplary biocompatible, intraluminal devices are currently produced by several companies including Cordis, Boston Scientific, Guidant, and Medtronic (Detailed description of currently available catheters, stents, wires, etc., are available at the websites of Cordis Corporation (cordis.com); Medtronic, Inc. (medtronic.com); and Boston Scientific Corporation (bostonscientific.com)).
  • the invention also provides articles of manufacture including pharmaceutical compositions of the invention and related kits.
  • the invention encompasses any type of article including a pharmaceutical composition of the invention, but the article of manufacture is typically a container, preferably bearing a label identifying the composition contained therein.
  • the container can be formed from any material that does not react with the contained composition and can have any shape or other feature that facilitates use of the composition for the intended application.
  • a container for a pharmaceutical composition of the invention intended for parental administration generally has a sterile access port, such as, for example, an intravenous solution bag or a vial having a stopper pierceable by an appropriate gauge injection needle.
  • Cell-based and/or non-cell-based compositions for use in the therapeutic methods of the present invention may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • the invention contemplates that
  • Optimal concentrations of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists.
  • biologically acceptable medium includes solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the one or more agents.
  • media for pharmaceutically active substances is known in the art. Except insofar as a conventional media or agent is incompatible with the activity of a particular agent or combination of agents, its use in the pharmaceutical preparation of the invention is contemplated.
  • Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations".
  • compositions of the present invention may be given orally, parenterally, or topically. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, ointment, controlled release device or patch, or infusion.
  • the effective amount or dosage level will depend upon a variety of factors including the activity of the particular compositions employed, the route of administration, the time of administration, the rate of excretion of the particular compositions being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the animal, and like factors well known in the medical arts.
  • compositions e.g., cell-based compositions alone or in combination with one or more non-cell based compositions
  • compositions can be administered as such or in admixtures with pharmaceutically acceptable and/or sterile carriers and can be administered concomitantly or concurrently with other compounds.
  • pharmaceutically acceptable compositions comprising an effective amount of cell or non-cell based compositions, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) delivery via a catheter, port or other biocompatible, intraluminal device; (2) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (3) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension.
  • the cells or cell clusters may be surgically or laproscopically implanted either near the pancreas or in the abdominal cavity, or at a distant and more accessible site.
  • the subject compositions may be simply dissolved or suspended in sterile water.
  • the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.
  • the pharmaceutically acceptable carrier materials include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene
  • compositions for administration may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium
  • the absorption of the agent in order to prolong the effect of an composition, it is desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the composition then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered composition form is accomplished by dissolving or suspending the agent in an oil vehicle.
  • the invention contemplates administration to neonatal, adolescent, and adult patients, and one of skill in the art can readily adapt the methods of administration and dosage described herein based on the age, health, size, and particular disease status of the patient. Furthermore, the invention contemplates administration in utero to treat conditions in an affected fetus.
  • the invention contemplates therapeutic methods comprising administration of pharmaceutical preparations concurrently or concomitantly with immuno-suppressants and/or other anti-rejection drugs.
  • the invention contemplates administration via the same or via a different mode of administration, and similarly contemplates that the compositions are each formulated appropriately in light of their properties and the desired route of administration.
  • the invention contemplates additional methods of preventing host rejection of the differentiated cells. Such methods are based on inducing tolerance in the patient, and can be used alone or in combination with other immunosuppresants or anti-rejection drugs.
  • tolerance is induced by first introducing into the patient dendritic cells differentiated from the same line of ES cells that will be used to differentiate the particular endodermal cells.
  • ES cells can be differentiated into dendritic cells by first driving them down the hematopoietic lineage via addition of one or more factors including, but not limited to, IL-I, IL-3, IL-6, GM-CSF, G-CSF, SCF, or erythropoietin.
  • the ES cells can be co-cultured with cell lines such as OP-9 stromal cells or yolk-sac endodermal cells. Following differentiation of ES cells to dendritic cells, essentially purified populations of dendritic cells can be prepared and delivered to the patient.
  • dendritic cells are delivered to the patient prior to administration of the therapeutic cells (e.g., the pancreatic cells or the hepatic cells).
  • the dendritic cells are optionally delivered along with traditional immunosuppressive therapies.
  • the therapeutic cells When the therapeutic cells are later delivered, they may optionally be delivered with the same or with a lower dose of immunosuppresants.
  • the invention contemplates that these partially or terminally differentiated adult stem cells can be used therapeutically in all of the ways described for embryonic stem cells.
  • adult stem cells potential graft rejection can be eliminated by using cells derived from the patient to be treated.
  • immunosuppressive and tolerance approaches are also contemplated.
  • Example 1 Human Embryonic Stem Cells Spontaneously Differentiate to Ectodermal, Mesodermal, and Endodermal Cell Types
  • Figure 1 confirms previous experiments demonstrating that human embryonic stem (ES) cells spontaneously differentiate along all three lineages when cultured as embryoid bodies (EBs).
  • Human embryonic stem cell lines 1 or 2 (hESl and hES2) were used to generate embryoid bodies. Briefly, ES cells were removed from the MEF feeder layer by either manual cutting (M) or collagenase digestion (C). The removed ES cells were then placed in appropriate media. After 0, 5, or 9 days post-EB formation, RNA was extracted from the EBs and analyzed for expression of the indicated markers by real-time RT-PCR. Relative expression shown is normalized to that of ⁇ -actin and expression for day 0 was set equal to 1. An asterisk (*) indicates arbitrary values due to no expression at day 0.
  • M manual cutting
  • C collagenase digestion
  • One method for directing the differentiation of stem cells is to generate embryoid bodies. These embryoid bodies can be grown under a number of conditions including, but not limited to, in floating suspension culture, in MATRIGELTM or other matrix, or on a filter. However, the first step is the actual formation of an embryoid body from a culture of embryonic stem cells. We used any of the following methods for generating embryoid bodies from cultures of embryonic stem cells. These methods can be used to generate embryoid bodies from human embryonic stem cells grown on MEF feeder layers, embryonic stem cells grown on other feeder layers, and embryonic stem cells grown under feeder free conditions.
  • Human embryonic stem cells e.g., lines hES 1-6 or DM lines
  • culture medium PBS
  • collagenase IV stock solution 5 mg/ml
  • trypsin/EDTA stock solution 0.25%
  • DM lines ultra-low-6-well plates.
  • the following protocol can be used to generate embryoid bodies. This protocol was specifically used to generate embryoid bodies from cell lines hES 1-6. However, the protocol can be used more generally in other ES cell or cell lines.
  • PlOO tissue culture plates containing hES cells grown under standard conditions were used as starting material. The medium was aspirated, and the cells were washed 2 times with PBS. After the wash, 3 ml of 1 mg/ml collagenase IV was added to each plate, and the plates were incubated for 8 minutes in a 37°C tissue culture incubator. Following incubation, the collagenase was aspirated from the cells, and the cells were washed with 10 ml of PBS. The PBS was gently aspirated, and care was taken to avoid disturbing the colonies of embryonic stem cells.
  • EB culture medium About 8 ml of EB culture medium was gently added to the plate, and the plate was mechanically and gently streaked using a 5-ml plastic pipette or a cell scraper. The materials were pipetted up and down to dislodge the cells pieces - care was taken not to over-pipette and damage the cells.
  • the embryonic stem cell clusters were transferred to ultra-low 6-well plates to promote embryoid body formation. Embryoid bodies were cultured for several days, and EB culture medium was changed every 2-3 days.
  • EBs were handled as follows: EBs were collected in a tube, and were allowed to either settle to the bottom of the tube, or were spun briefly to facilitate precipitation of the EBs to the bottom of the tube. At this point, EBs can be processed for immunohistochemistry studies or for RNA extraction, using standard techniques.
  • the following protocol can be used to generate embryoid bodies. This protocol was specifically used to generate embryoid bodies from cell lines hES 1-6. However, the protocol can be used more generally in other ES cell or cell lines. Organ cultures dishes containing KES cells grown under standard conditions on MEF feeder layers were used as starting material. The medium was aspirated, and the cells were washed 2 times with PBS. Then 0.5 ml of 1 mg/ml collagenase IV was added to each dish, and the plates were incubated for 5 minutes in a 37°C tissue culture incubator. Following incubation, the collagenase was aspirated from the cells, and the cells were washed with 1 ml of PBS. The PBS was gently aspirated, and care was taken to avoid disturbing the colonies of embryonic stem cells.
  • EB culture medium was gently added to the plate. ES cell colonies were dissociated gently using a pipet tip. Care was taken to avoid detaching MEFs from the dish. ES cell pieces were transferred to a suspension plate containing EB culture medium to promote embryoid body formation. Embryoid bodies were cultured for several days, and EB culture medium was changed every 2-3 days.
  • EBs were handled as follows: EBs were collected in a tube, and were allowed to either settle to the bottom of the tube, or were spun briefly to facilitate precipitation of the EBs to the bottom of the tube. At this point, EBs can be processed for immunohistochemistry studies or for PxNA extraction, using standard techniques.
  • the following protocol can be used to generate embryoid bodies. This protocol was specifically used to generate embryoid bodies from the Harvard cell line HUES-I. However, the protocol can be used more generally in other ES cell or cell lines.
  • PlOO tissue culture plates containing hES cells grown under standard conditions on MEF feeder layers were used as starting material. The process begins with a stage whereby the ES cells were waned from the feeder layer. The cells were trypsinized with 0.05% trypsin and plated on collagen IV-coated plates in the splitting ratio of 1 :3. The cells were cultured for 3-4 until subconfluent.
  • the medium was aspirated, and the cells were washed 2 times with PBS.
  • About 3 ml of 1 mg/ml collagenase IV was added to each dish, and the plates were incubated for 4 minutes in a 37°C tissue culture incubator.
  • the collagenase was aspirated from the cells, and the cells were washed with 10 ml of PBS.
  • the PBS was gently aspirated, and care was taken to avoid disturbing the colonies of embryonic stem cells.
  • EB culture medium About 8 ml of EB culture medium was gently added to the plate, and the plate was mechanically scraped using a 5-ml plastic pipette and cell scraper. The materials were pipetted up and down to dislodge the cells pieces - care was taken not to over-pipette and damage the cells.
  • the embryonic stem cell clusters were transferred to suspension plates to promote embryoid body formation. Embryoid bodies were cultured for several days, and EB culture medium was changed every 2- 3 days.
  • EBs were handled as follows: EBs were collected in a tube, and were allowed to either settle to the bottom of the tube, or were spun briefly to facilitate precipitation of the EBs to the bottom of the tube. At this point, EBs can be processed for immunohistochemistry studies or for RNA extraction, using standard techniques.
  • Example 3 Method for Directing the Differentiation of a Stem Cell to a Particular Differentiated Cell Type
  • the following is indicative of protocols that can be used to direct the differentiation of stem cells to a particular differentiated cell type.
  • the particular protocol outlined here promoted differentiation of embryonic stem cells along the pancreatic lineage, as assayed by expression of the marker pdx-1.
  • Materials Human embryonic stem cells; medium (RPMI / 20% serum replacement (20SR) / pen-strep; PBS; collagenase IV (preferably for use with hES 1-6 lines); trypsin / EDTA (preferably for DM lines); ultra-low attachment-6-well plates (Corning / costar) growth factor reduced MATRIGELTM; early factors (EF); late factors (LF).
  • DO in the time table provided above indicates the point at which culture of cells as embryoid bodies begin.
  • DO indicates the point at which the embryonic stem cells are plated directly onto MATRIGELTM or other tissue culture plates.
  • cultures of proliferating ES cells Prior to DO, cultures of proliferating ES cells must be handled according to one of the protocol outlined above to generate a starting culture of EBs.
  • PlOO tissue culture plates containing hES cells grown under standard conditions were used as starting material.
  • the medium was aspirated, and the cells were washed 2 times with PBS.
  • About 3 ml of 1 mg/ml collagenase IV was added to each plate, and the plates were incubated for 8 minutes in a 37°C tissue culture incubator.
  • the collagenase was aspirated from the cells, and the cells were washed with 10 ml of PBS.
  • the PBS was gently aspirated, and care was taken to avoid disturbing the colonies of embryonic stem cells.
  • EB culture medium RPMI/20SR
  • RPMI/20SR EB culture medium
  • the materials were pipetted up and down to dislodge the cells pieces - care was taken not to over-pipette and damage the cells.
  • the pellets were transferred to a 15-ml tube, and the pellets were spun down at 2500 rpm for 4 minutes. The medium was aspirated.
  • MATRIGELTM medium e.g., 1 ml liquefied MATRIGELTM + 5 ml RPMI/20SR
  • MATRIGELTM medium e.g., 1 ml liquefied MATRIGELTM + 5 ml RPMI/20SR
  • Total volume is according to 2 ml for each well.
  • the hES cell pellets were resuspended in the MATRIGELTM medium, and 2 ml of hES pelletmedium suspension was added / well of Ultra-low plate.
  • factor cocktail 100 ng each can either be added in the MATRIGELTM medium before pellet resuspension procedure or immediately after suspension is plated in the Ultra-low plate. Plates were incubated in 37 0 C tissue culture incubator, and the MATRIGELTM medium gels after several hours. After overnight incubation, the hES pellets formed embedded embryoid bodies.
  • the medium was removed. Care was taken to prevent dislodgement of the EBs and the MATRIGELTM. Fresh medium was added to the cells which were allowed to equilibrate for approximately 1 hour. This was followed by the addition of the LF cocktail and RPMI/20SR.
  • the MATRIGELTM EBs were collected into a 15 ml FALCON tube. 12 ml chilled PBS was added, and the EBs were placed on ice for 10 min. The EBs were spun for 4 minutes at 2500 rpm (round per minute). The supernatant was carefully removed, and the EBs were transferred to an EPPENDORF tube. The EBs were then analyzed using immunocytochemistry or RT-PCR.
  • FIG. 2 provides a schematic representation of a multi-step method for directing the differentiation of stem cells along particular endodermal lineages.
  • the starting material is embryonic stem cells
  • the particular endodermal lineage is pancreatic - specifically beta islet cells.
  • Embryonic stem cells can be cultured in any of a number of formats, for example, as embryoid bodies in suspension culture, as embryoid bodies embedded in MATRIGELTM or other matrix material, as embryoid bodies on a filter, as embryonic stem cells directly plated on MATRIGELTM or other matrix, or as embryonic stem cells directly plated on tissue culture plates.
  • embryonic cells cultured in any of the foregoing ways are cultured for 1-10 days (or even 1-15 days) in medium containing early factors. This period of culture directs the cells down a particular endodermal pathway.
  • culture in early factors results in partial differentiation, as assessed by expression of the early marker Pdx-1.
  • Exemplary early factors that help to induce the expression of pdx-1 include, but are not limited to, activin A, BMP2, BMP4, and nodal. Such factors can be added individually or in combination. Combinations include combinations of two, three, four, or more than four factors.
  • cells are cultured for 1-10 days (or even 1-15 days) in medium supplemented with late factors. This period of culture further promotes differentiation of cells along the particular pathway toward terminal differentiation. This may include promotion of further expression of pdx-1, promotion of expression of terminal markers of differentiation, both promotion of further expression of pdx-1 and further expression of insulin, or decrease expression of pdx-1 accompanied by an increased expression of markers of terminal differentiation.
  • pancreatic cell types and specifically for beta islet cells, culture in late factors promotes further differentiation. Promotion of further differentiation can be assessed by assaying for a further increase in expression of pdx-1. Additionally or alternatively, further differentiation can be assayed by expression of late markers including insulin. Note thai pdx-1 expression is maintained, although perhaps at a lower level, upon terminal differentiation of the cells.
  • Exemplary late factors that help induce expression of markers of terminal beta islet differentiation include, but are not limited to, HGF, exendin4, betacellulin, and nicotinamide. Such factors can be added individually or in combination. Combinations include combinations of two, three, four, or more than four factors. At this point, cells may optionally be further cultured to enhance terminal differentiation and functional performance.
  • Human embryonic stem cell 3 (hES3) were subjected to a multi-step differentiation protocol, as outlined in Figure 2.
  • the embryonic stem cells were cultured as embryoid bodies suspended in 3D in MATRIGELTM.
  • the cells were cultured for 10 days in medium containing the early factors and then for 10 days in medium containing the late factors. Following culture, cells were assayed for expression of pdx-1.
  • Figure 3 summarizes the results of these experiments.
  • the embryoid bodies were cultured, except as indicated, with the following early and late factors: early factors were activin A, BMP2, BMP4, and nodal; late factors were HGF, exendin4, betacellulin, and nicotinamide. The particular factor omitted is indicated under each bar.
  • FIG. 4A shows a cluster of cells expressing ⁇ -galactosidase, thus indicating expression of pdx-1, after EB formation and subsequent plating.
  • Figure 4B shows quantitative RT-PCR data for pdx-1 for mouse EBs at various stages of culture. It is apparent that pdx-1 expression increased over time up to 24 days of EB formation.
  • Example 7 Directed Differentiation Along a Pancreatic Lineage Increases Over Time
  • FIG. 5A shows that pdx-1 mRNA, as detected by real time RT-PCR, increased with the number of days of EB formation between 0 to 24 days.
  • Figure 5B shows an ethidium bromide stained gel of the pdx-1 RT-PCR product, indicating that a single band of the predicted size was detected.
  • Example 8 Various Growth Factor Preparations Promote Directed Differentiation Along a Pancreatic Lineage
  • Human embryonic stem cell line hES3 was used to generate EBs suspended in MATRIGELTM. Addition of TGF- ⁇ factors increased pdx-1 expression in hES3. hES3 EBs were cultured in MATRIGELTM in RPMI media with addition of several TGF- ⁇ related factors exemplified by Activin, BMP-2, BMP-4 or Nodal. Expression of pdx-1 by RT-PCR was measure after 20 days in culture. As shown in Figure 6, expression is expressed as fg per ng actin. Addition of growth factors led to a 9-fold increase in pdx-1 expression in comparison to culture in the absence of growth factors. Furthermore, this treatment resulted in an increase in insulin expression, as measured by RT-PCR, after 20 days in culture. Expression is expressed as fg per ng actin. Addition of growth factors led to about a 2-fold increase in insulin expression.
  • Example 9 Directed Differentiation of Human Embryonic Stem Cells Along Particular Endodermal Lineages
  • the methods of the invention can be used to direct the differentiation of stem cells to particular endodermal cell types.
  • the results summarized in Figure 7 demonstrate that hepatocyte cell types can be differentiated from embryonic stem cells.
  • Figure 7 A shows expression of the hepatocyte marker albumin in hES cells cultured by directly plating ES cells on MATRIGELTM coated tissue culture plates.
  • Figure 7B shows quantitative RT-PCR data for two different hES lines subjected to several differentiation protocols.
  • Human ES cells differentiated according to condition E had the largest increase in expression of several hepatic markers.
  • ES cells were plated on MATRIGELTM coated plates and maintained in knock-out media supplemented with 20% serum replacement and 1% DMSO. After five days, cells were treated with 2.5 mM sodium butyrate. The medium was then replaced with hES media supplemented with 100 ng/ml alpha-FGF, 0.1 ng/ml TGF- ⁇ , 30 ng/ml EGF, and 30 ng/ml HGF. Finally, the medium was replaced with hES media supplemented with 10 ng/ml oncostatin M, 1 ⁇ M dexamethasone, 30 ng/ml HGF, and 0.1 ng/ml TGF- ⁇ .
  • Example 10 Directed Differentiation of Human Embryonic Stem Cells Along a Pancreatic Lineage Using a Combinatorial Approach - 44 day protocol
  • the differentiation of human embryonic stem cells grown in 3-dimensional culture can be directed along the pancreatic lineage, as indicated by expression of pdx-1. Additional experiments were then conducted to see whether the directed differentiation of human embryonic stem cells along pancreatic lineage can be further influenced by subjecting the cells to a combination of the 3-D culture system outlined above and other culture systems shown by us to influence differentiation of cells to pancreatic cell types.
  • human embryonic stem cells were differentiated, as described above, in 3-dimensional culture in MATRIGELTM for 20 days. For the first 10 days the cells were cultured in the presence of early factors (Activin A: 50 ng/ml; BMP2: 50 ng/ml; BMP4: 50 ng/ml; Nodal: 50 ng/ml) and for the second 10 days the cells were cultured in the presence of late factors (Betacellulin: 50 ng/ml; HGF: 50 ng/ml; Exendin-4: 10 ng/ml, Nicotinamide: 10 mM).
  • early factors Activin A: 50 ng/ml
  • BMP2 50 ng/ml
  • BMP4 50 ng/ml
  • Nodal 50 ng/ml
  • Betacellulin 50 ng/ml
  • HGF 50 ng/ml
  • Exendin-4 10 ng/ml
  • Nicotinamide 10 mM
  • pdx-1 levels were measured by RT-PCR at various time points during culture of the cells in the 3D MATRIGELTM protocol. RNA was isolated and analyzed for pdx-1 and actin expression by RT-PCR. Data were standardized in comparison to actin expression, and the results are expressed as absolute g pdx-1 1 kg Actin expression +/- SD. These results are summarized in Figure 8.
  • DO-IO Days 0-10): Day 0 is normally the set up day.
  • Cells were cultured in KO SR medium + early growth factor cocktail (Activin A: 50 ng/ml; BMP2: 50 ng/ml; BMP4: 50 ng/ml; Nodal: 50 ng/ml).
  • the medium was topped up (to feed the cells) at D 3, 6.
  • D 10-20 (Days 10-20): Cells were cultured in KO SR medium + late growth factor cocktail (Betacellulin: 50 ng/ml; HGF: 50 ng/ml; Exendin-4: 10 ng/ml; Nicotinamide: 10 mM). The medium was changed at 16.
  • D26 (Day 26): EBs were dissociated by Dispase (1 of 2 wells, the other one remains as EB) and re-plated on low attachment plates.
  • D26-32 (Days 26-32): Step 2 of 24 day protocol.
  • Cells were cultured in basal medium + 20 ng/ml FGF- 18, 2 ⁇ g/ml Heparin (new medium at D26, new GFs added at D29).
  • D32-36 (Days 32-36): Step 3 of 24 day protocol.
  • Cells were cultured in basal medium + 20 ng/ml FGF-18, 2 ⁇ g/ml Heparin, 10 ng/ml EGF, 4 ng/ml TGF ⁇ , 30 ng/ml IGFI, 30 ng/ml IGFII, 10 ng/ml VEGF (new medium at D32, new GFs added at D34).
  • D36 (Day 36): Cells were re-plated on Fibronectin-coated plates. D36-40 (Days 36-40): Step 4 of 24 day protocol. Cells were cultured in
  • RPMI medium 11 mM GIc, 5% FBS, 2 mM Glutamax, 8 mM HEPES, Penicillin/Streptomycin + 10 ⁇ M Forskolin, 40 ng/ml HGF, 200 ng/ml PYY (new medium at D36, new GFs added at D38).
  • D40-44 (Day 40-44): Step 5 of 24 day protocol.
  • Cells were cultured in CMRL medium (5 mM GIc, 5% FBS, 2 mM Glutamax, Penicillin/Streptomycin) + 100 ng/ml Exendin-4, 5 mM Nicotinamide (new medium at D40, new GFs added at D42).
  • D44 (Day 44).
  • RNA was harvested for analysis of pdx-1 and insulin expression by RT-PCR.
  • RNA samples were harvested from cells at various points along this process to help evaluate the directed differentiation of the cells.
  • embryonic stem cells differentiate to express a high level of pdx-1.
  • FIG 9 A cells subject to both the 20 day 3D differentiation protocol and the 24 day protocol continue to express pdx-1.
  • pdx-1 expression is at a lower level than following the first 20 days in culture (compare Figure 8 and Figure 9A). Additionally, however, these cells express very high levels of insulin mRNA (See, Figure 9B). Expression of insulin protein will be confirmed by performing a C- peptide ELISA assay.
  • Example 11 Directed Differentiation of Human Embryonic Stem Cells Along a Pancreatic Lineage Using a Combinatorial Approach - 34 Day protocol
  • the differentiation of human embryonic stem cells grown in 3-dimensional culture can be directed along the pancreatic lineage, as indicated by expression of pdx-1. Additional experiments were then conducted to see whether the directed differentiation of human embryonic stem cells along the pancreatic lineage can be further influenced by subjecting the cells to a combination of the 3D culture system outlined above and other culture systems shown by us to influence differentiation of stem cells.
  • One such other culture system was the 24 day protocol outlined in Example 10.
  • DlO Day 10: EBs were eluted from 3D MATRIGELTM and re-plated on low attachment plates.
  • D10-18 Day 2 of 34 day protocol. Cells were cultured in basal medium ((DMEM / F12, 17 nM Glucose, 2 nM Glutamine, 8 mM HEPES, 2% B27 and Pen / strep) + 20 ng/ml FGF-18, 2 ⁇ g/ml Heparin, The cells were fed on day 13 and 16 with media plus growth factor top ups.
  • basal medium (DMEM / F12, 17 nM Glucose, 2 nM Glutamine, 8 mM HEPES, 2% B27 and Pen / strep) + 20 ng/ml FGF-18, 2 ⁇ g/ml Heparin
  • D18-24 (Days 18-24): Step 3 of 34 day protocol.
  • Cells were cultured in basal medium + 20 ng/ml FGF-18, 2 ⁇ g/ml Heparin, 10 ng/ml EGF, 4 ng/ml TGF ⁇ , 30 ng/ml IGFI, 30 ng/ml IGFII, 10 ng/ml VEGF.
  • the cells were fed fresh medium and growth factors on Day 21.
  • D24-29 (Days 24-29): Step 4 of 34 day protocol.
  • Cells were cultured in RPMI medium (11 mM GIc, 5% FBS, 2 mM Glutamax, 8 mM HEPES,
  • D29-34 (Days 29-34): Step 5 of 32 day protocol.
  • Cells were cultured in CMRL medium (5 mM GIc, 5% FBS, 2 mM Glutamax, Penicillin/Streptomycin) + 100 ng/ml Exendin-4, 5 mM Nicotinamide. The cells were fed fresh medium and growth factors on Day 32.
  • D34 Continue culturing cells in CMRL w/o growth factors and harvest the cells.
  • RNA samples were harvested from cells at various points along this process to help evaluate the directed differentiation of the cells. Furthermore, culture medium and factors were regularly changed throughout the differentiation protocol. Results: As outlined in Example 10 above, following the standard 3D differentiation protocol, embryonic stem cells differentiate to express a high level of pdx-1. We additionally directed the differentiation of embryonic stem cells along a pancreatic lineage using the approach outlined in Example 11. During the protocol outlined in Example 11, we harvested samples at various points during the differentiation protocol. Figure 10 summarizes the results for two time points: day 10 (just prior to beginning the 34 day protocol) and day 22 (approximately 1/3 of the way through the 34 day differentiation protocol).
  • Figures 1OA and 1OB show that at day 10 (prior to beginning the 34 day protocol), expression of pdx-1 is low and expression of insulin is undetectable. However, as shown in Figures 1OA and 1OB, at 22 days the expression of pdx-1 is very high. In fact, expression of pdx-1 at this point is higher than after completion of the 24 day protocol (see, Figure 9A). As shown in Figure 1OB, at 22 days insulin expression can be detected. However, expression of insulin at this point is not as robust as following the completion of the 24-day protocol (see, Figure 9B). This may indicate that part way through the 34-day differentiation protocol, the cells are continuing to terminally differentiate along the pancreatic lineage. At approximately 22 days, expression of pdx-1 is still relatively high, perhaps indicating that more cells are capable of but have not yet differentiated to insulin expressing cells.
  • Analysis of cells using similar methods at various time points in the 34-day differentiation protocol can refine the time and conditions under which cells are directed to partially versus terminally differentiate along a pancreatic lineage.
  • Example 12 Directed Differentiation of Embryonic Stem Cells to PdX-I + Cells Using the 20-Day Differentiation Protocol Mimics Normal Pancreatic Development
  • Mimics Normal Pancreatic Development Much of the work aimed at the in vitro generation of beta cells from embryonic and adult stem cells has focused on the activation of the pdx-1 gene due to its tissue-specific expression in early pancreatic progenitors.
  • pdx-1 expression is aimed at the in vitro generation of beta cells from embryonic and adult stem cells.
  • the particular in vitro differentiation scheme used may result in spurious activation of pdx-1.
  • initiation protocol that directs the differentiation of embryonic stem cells along a pancreatic lineage.
  • EF:LF early/late factor
  • This 2 EF-3 LF protocol included the early factors Activin A (50 ng/ml) and BMP -4 (50 ng/ml). The cells were cultured in the early factors as previously described from day 0 to day 10.
  • the 2 EF-3 LF protocol included the late factors HGF (50 ng/ml), exendin-4 (10 ng/ml), and ⁇ -cellulin (50 ng/ml).
  • the 2 EF-3 LF protocol represents an improvement over the previous 4 EF-4 LF protocol because it induced robust pdx-1 expression using a cheaper, faster, and simpler procedure.
  • Figures 13-16 summarize the experiments that led to the development of the 2 EF-3 LF protocol. Without being bound by theory, since BMP2 and BMP4 are two structurally- related growth factors that bind the same cell surface receptor complex, it was anticipated that the combination of the two growth factors was super-saturating in the 4 EF mix and that one factor alone would be sufficient for pdx-1 induction.
  • Figure 13 A summarizes data showing that an EF growth cocktail lacking BMP2 induced pdx-1 expression at levels slightly greater than that induced by all four early EFs (compare G2 to G7 in Figure 13A).
  • TGF- ⁇ -related ligand Nodal which is an evolutionarily conserved endoderm inducer, had little effect in the EF cocktail.
  • "AU" indicates the addition of all four of the EF and/or LF used in the initial 4 EF-4 LF 20-day protocol. Duplicate wells for each experimental condition are shown along with Ct value Pdx-1 / actin of the best performing conditions. Note that the results depicted in Figure 13 A represent normalized expression, and the resulted depicted in Fig. 13B represent Pdx-1 expression as % actin input.
  • MATRIGELTM low levels of pdx-1 are first detected as early as 12 days (See, for example, Figures 4B, 11, 12) and increase gradually over time.
  • the growth factors, HGF, exendin-4 and beta-cellulin have been extensively characterized for their roles in the maturation, proliferation or modulation of the insulin-secreting kinetics of more specialized islet-derived cell populations, and are thus predicted to provide little instructive signaling to the emerging pdx-1 -expressing pancreatic progenitors.
  • the contribution of each of the 3 LF as well as nicotinamide toward the expression of pdx-1 on day 20 was assessed.
  • Figure 17 shows immuno-localization of Pdx-1 in embryoid bodies differentiated using the initiation protocol.
  • EBs were differentiated for 20 days using either the 4 EF-4 LF protocol ( Figures 17A and 17B) or the 2 EF-3 LF protocol ( Figures 17C and 17D).
  • initiation protocol Pdx-1 -positive cell populations were identified within epithelial ribbons that often enclose lumens and are often confined to the EB periphery (arrows in Figures 17A and 17B).
  • the clusters of Pdx-1 -expressing cells may suggest that a subpopulation of EBs support a pancreatic "niche" that underlies the further development of insulin-producing cells.
  • Figure 22 The expression of pdx-1 mRNA was further analyzed by in situ hybridization (Figure 22), and shown to precisely correlate with Pdx-1 immunohistochemistry.
  • Figures 22 A and 22B show pdx-1 expression by in situ hybridization after the 20-day initiation protocol (2 EF-3 LF).
  • the results summarized in Figures 22A and 22B indicated that approximately 1/3 of all EBs harvested from an individual culture well express Pdx-1 (darker staining) after 20 days of differentiation.
  • the higher magnification view shown in Figure 22B demonstrates that pdx-1 transcripts localized near the periphery of the EB.
  • Figure 22C indicated that EBs cultured in the absence of growth factors fail to express Pdx-1.
  • Figure 22D summarizes the results of quantitative PCR of parallel cultures of those cells shown in Figures 22A and 22C, and confirms robust pdx-1 expression in cultures containing growth factors (higher bar) versus those differentiated in the absence of growth factor (lower bar).
  • the expression pattern of pdx-1 in the EBs was further investigated by exploring whether Pdx-1 and C-peptide expressions co-localize.
  • the hES3 cells were directed to differentiate using 2EF-3LF initiation protocol, and a simplified maturation protocol using simply Step 4, as further described in Example 15, "Step- 4 only maturation.”
  • Sections of the resulting EBs were prepared and immuno- stained using antibodies to Pdx-1 and C-peptide, either as a single or double stained immunohistochemistry samples.
  • the results are shown as Figure 28, wherein the top panels show the high magnification images and the bottom panels show lower magnification images, with DAPI-stained nuclei.
  • Figure 28 shows that Pdx-1- positive cells localized in clusters or epithelial ribbons of the differentiated hES3 cells, and there are C-peptide positive cells among them.
  • the initiation protocols provide a straightforward differentiation regime that directs pluripotent embryonic stem cells toward pdx-1 -expressing pancreatic progenitors.
  • insulin expression is still relatively low.
  • Figure 18A provides a schematic representation of a particular combination of the 2 EF-3 LF initiation protocol plus a maturation protocol.
  • the maturation protocol depicted in Figure 18 A may have as many as 5 steps. The use of any combination of these steps will be referred to as a maturation protocol, and reference to the stage/step, the number of days in culture, and/or the particular factors used will distinguish permutations of the maturation protocol.
  • the baseline concentration of C-peptide detected after 48 hours of culture was approximately 0.5 ng/ml, as measured in control cultures that have progressed through the first four steps of the maturation protocol - upper schematics - Figures 18A and 19 A.
  • Figure 19B summarizes the results of these experiments. Interestingly, there was little difference in C-peptide release on day 36 for each of the other conditions aside from Stage 4, which showed a 6-fold increase in C-peptide release (Figure 19B). There are many unique aspects to this particular stage: most notably is the continued use of RPMI as the base media, and growth on fibronectin-coated dishes.
  • Step-4 only maturation The maturation protocol was further refined as shown in the diagrams of
  • FIG. 23 The top diagram is the original 5 step maturation protocol.
  • the middle diagram shows using only Step 4 of the 5 -step protocol.
  • Twenty-day old MATRIGELTM EBs were washed with cold PBS to remove excess MATRIGELTM, and replated onto fibronectin-coated dishes and cultured directly in Step 4 medium for 4 days. Steps 1-3, as well as Step 5 were omitted.
  • Step 4 of the muti-step protocol is the key step to the release of C-peptide from differentiating hES3 cells.
  • Figure 24 various permutations of the maturation steps were investigated.
  • the arrows at the bottom of the graph show time points when the culture medium was either changed or maintained, according to the permutations shown at the top of the graph.
  • Step 4 medium was modified by removing some of the components, and conditioned medium was collected on day 30 from cultures grown in the modified Step 4 medium.
  • Step 4 medium is based on RPMI and normally contains 5% FBS, 10 ⁇ g forskolin, 40 ng/ml HGF, and 200 ng/ml PYY.
  • the result of the experiment is shown in Figure 25.
  • the removal of all additional growth factors and forskolin, while retaining 5% FBS, causes an approximately two-fold decrease in the levels of C-peptide in the culture medium.
  • Step 4 medium was prepared using either RPMI following the standard protocol or CMRL, supplemented with FBS, a commercially available serum replacer (SR) which is chemically defined, or not supplemented at all.
  • SR serum replacer
  • C-peptide levels were measured on Day 28.
  • Figure 26A there was no statistically significant difference in C-peptide release between culture using medium based on RPMI or CMRL.
  • FBS center bar
  • SR right bar
  • hES3 cells were differentiated using the simplified maturation protocol described above. On day 30 of the protocol, the culture medium was removed and replaced by either RPMI or DMEM supplemented by 22 rnM glucose, RPMI supplemented with 22 niM glucose, or DMEM supplemented by 5 niM glucose. 5 mM glucose mirrors the physiological concentrations of glucose at which insulin is stockpiled in secretory granules.
  • WISH whole-mount in situ hybridization
  • Figure 21 shows paraffin sections of day 45 EBs following immunocytochemistry with C-peptide.
  • C-peptide-positive cells were often distributed at the EB periphery in a manner similar to insulin- expressing cell clusters, but were sometimes located in more interior regions (Figure 21B-F).
  • Figure 21 A shows, as a positive control, C-peptide immunolocalization in an adult mouse islet.
  • Similar co-localization can be seen when the EBs subject to the simplified protocol without Steps 1 and 5 were immunofluorescent stained.
  • Figure 32 shows high magnification (top panels) and low magnification (bottom panels) images of paraffin-embedded and sectioned EBs harvested on Day 26 from the simplified protocol. At this stage, most insulin positive cells were also reactive for C-peptide, which is the by-product of insulin synthesis. Nuclei are shown by the staining by DAPI.
  • Figure 33 shows additional evidence of differentiation through the simplified protocol without standard protocol Steps (1) and (5).
  • the immunofluorescent stain showed efficient formation of cells belonging to the beta- cell lineage (left panels: top, high magnification; bottom, low magnification). These cells were largely confined to epithelial ribbons or tubes that enclose luminal spaces.
  • Pdx-1 -positive cells clusters are also reactive for glucose transporter Glut2, as can be seen in the right panels. Nuclei are shown by the staining by DAPI.
  • hES cells were cultured according to standard procedures. The generation of embryoid bodies (EBs), and the culture of the cells within the 3D MATRIGELTM during the first 20 days were as follows:
  • Pre-chill 2 ml, 5 ml and 10 ml pipettes at - 2O 0 C.
  • MATRIGELTM medium e.g., 1 ml liquefied MATRIGELTM + 5 ml RPMI/20SR
  • Total volume is according to 2 ml for each well.
  • One PlOO plate of hES colonies can be split into 3 to 5 wells depending on the confluence.
  • growth factor cocktail 100 ng Activin A + 100 ng BMP4 per well
  • growth factor cocktail 100 ng Activin A + 100 ng BMP4 per well
  • Late factor stage DlO Remove medium with 1-ml blue pipette under dissecting microscope very slowly and carefully. Do not suck away MATRIGELTM and EBs.
  • RNA extraction Use high speed to get EB pellet. (Refer to Qiagen kit protocol or Trizol protocol.)
  • RLT lysate or Trizol lysate is better to be kept at - 70°C for at least a few hours before extraction. This freeze and thaw cycle appears to help lysing.
  • Trizol method gives more than double amount higher of the final RNA yield.
  • DNA shredding step needs to be thorough and DNAase treatment step is preferred to be 1 hour in order to get rid of the genomic DNA cleanly.
  • Culture conditions beyond the initial 20 day protocol, in which pdx-1- expressing cells were directed toward more mature insulin producing cell populations, will be referred to as the maturation procedure(s).
  • EBs were removed from their RPMI- MATRIGELTM cultures by centrifugation and cold PBS washes, and transferred to a basal medium (DMEM/F12, 17 mM glucose (GIc), 2 mM glutamax, 8 mM HEPES, 2% B27 supplement, and 1 x pen/strep) thought to promote the recovery and survival of beta cells.
  • a basal medium DMEM/F12, 17 mM glucose (GIc), 2 mM glutamax, 8 mM HEPES, 2% B27 supplement, and 1 x pen/strep
  • the cells were cultured for a further 6 days in the same basal media supplemented with 20 ng/ml FGF-18 and 2 ⁇ g/ml heparin.
  • the cells were cultured in basal media supplemented with FGF- 18 (20 ng/ml), heparin (2 ⁇ g/ml), EGF (10 ng/ml), TGF ⁇ (4 ng/ml), IGF-I (30 ng/ml), IGF-II (30 ng/ml) and VEGF (10 ng/ml).
  • fibronectin-coated (10 ⁇ g/ml) tissue culture plates in a new media mix (RPMI + glucose (11 niM), FBS (5%), glutamax (2 mM), HEPES (8 mM), 1 x pen/strep, forskolin (10 ⁇ M), HGF (40 ng/ml), and PYY (200 ng/ml)).
  • the final stage (days 40-44) consisted of culture in a CMRL-based media (supplemented with glucose (5 mM), glutamax (2 mM), pen/strep (Ix), exendin-4 (100 ng/ml) and nicotinamide (5 mM).
  • CMRL-based media supplemented with glucose (5 mM), glutamax (2 mM), pen/strep (Ix), exendin-4 (100 ng/ml) and nicotinamide (5 mM).
  • hES cells (preferentially hES3) are digested by Collagenase (2 x pi 00 confluent dishes to one 6 well dish).
  • Plating Cells are first distributed to a 6-well low-attachment plate in MATRIGELTM to start the 3D culture, according to QC "MATRIGELTM EB
  • RNA from one well at day 20 of the 3D MATRIGELTM protocol (A) collect RNA from one well at day 20 of the 3D MATRIGELTM protocol; (B) collect supernatant before and 24 h after each medium change from start of the multi-step maturation protocol (10 ⁇ l are required for one ELISA well, take 100 ⁇ l medium and keep at -20), (C) collect RNA from wells at day 45.
  • Day 10 Medium Change; remove medium with 1 ml pipette under the dissecting microscope very slow and carefully. Do not suck away MATRIGELTM cells. Give 3 ml of RPMI-SR, equilibrate 1 hr in the incubator, then remove again and give new medium with late GFs.
  • step 2 Multi-step Maturation Protocol - D26-D32 2 ml/well basal medium + 20 ng/ml FGF- 18, 2 ⁇ g/ml Heparin Medium change at D29 (spin cells at 600 g for 4 min. in swing bucket, give new medium).
  • step 3 Multi-step Maturation Protocol - D32-D36
  • D36 - replating on Fibronectin coat 6-well plates for 1 hr with 10 ⁇ g/ml Fibronectin in PBS, wash 2 x with RPMI-SR, plate cells in new medium on coated plates.
  • step 4 Multi-step Maturation Protocol - D36-D40 2 ml/well new RPMI medium: RPMI + 11 mM GIc, 5% FBS, 2 mM
  • HGF 200 ng/ml PYY.
  • Fibronectin at 600 g for 4 min. in swing bucket, while doing that give 1 ml of new medium on the attached cells to avoid drying out, give recovered cells in new medium).
  • Embryoid bodies were isolated from the 3D MATRIGELTM by transferring the entire culture to a 15 ml falcon tube and chilling on ice.
  • the EBs were pelleted by centrifugation in a swinging-bucket rotor (1500 RPM). The cell pellet was then rinsed twice in ice-cold PBS and centrifuged in a similar fashion to remove residual MATRIGELTM.
  • EBs were fixed in 4% paraformaldehyde for 4 hours, rinsed twice in PBS, and then stored at 4 0 C in 70% ethanol.
  • EBs were then embedded in paraffin using standard dehydration / clearing / paraffin-embedding protocols with a Leica TP 1020 automated tissue processor (2 x 1 hr each in 70%, 95%, 100% ethanol followed by 2 x 1 hr in xylenes and 4 x 1 hr in paraffin). Sections were cut at 5 ⁇ m, and stored long-term at 4°C for eventual immunohistochemistry or in situ hybridizations. Pancreas tissue was similarly prepared but with extended fixation.
  • Immunohistochemistry Immunostaming was performed according to standard protocols using the vectorshield colorometric (DAB) detection kit. Slides were first de- waxed 2 x 10 min. in xylene, then hydrated in a standard ethanol series (100%, 95%, 90%, 70% ethanol, then 2 times in PBS). For antigen retrieval, slides were slowly heated in 10 mM sodium citrate solution to 95 °C (approximately 9 minutes on the defrost setting of a conventional microwave). The slides were then slowly cooled to room temperature (about 30 minutes) and rinsed twice in PBS.
  • DAB vectorshield colorometric
  • Endogenous peroxidase activity was quenched with a 20 min incubation in 3% H 2 O 2 followed by 2 rinses in PBS.
  • the slides were blocked for 1 hr in PBS containing 1% BSA and 5% serum (corresponding to the species from which the secondary antibody was derived).
  • the primary antibodies were diluted to the following concentrations: rabbit anti-Pdx-1 (1:30,000), guinea pig anti Pdx-1 (1 :2000), and goat anti-Pdx-1 (1:40,000).
  • rabbit anti-human C- peptide (Linco Research, lot#81(lP)) antibodies were used at a 1:5000 dilution. Slides were incubated with the diluted primary antibody overnight.
  • the slides were rinsed twice in PBS, and a biotinylated goat-anti-rabbit secondary antibody was applied for 1 hr at room temperature.
  • the ABC mixture (Vectashields) was placed on the sections (prepared by mixing 20 ⁇ l/ml reagent A and 20 ⁇ l/ml reagent B in PBS) for 30 min. then rinsed away with 3x PBS washes followed by addition of the DAB substrate (Vector Laboratories, SK-4100).
  • the color reaction was monitored closely by microscopy, and was stopped by dipping the slide in water and then rinsing once in 1 x PBS.
  • the sections were then dehydrated and cleared in xylenes (Sigma) using standard protocols before mounting in a xylene-based permanent mounting media (DPX neutral mounting media, Sigma).
  • RNA isolation, cDNA synthesis, and quantitative PCR Total RNA from hESC or EBs at various stages of differentiation was isolated using the Qiagen RNeasy kit or prepared using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA was and quantified by UV absorption. 1 to 5 ⁇ g of RNA was DNase I treated and converted to cDNA using M-MuLV reverse transcriptase (New England Biolabs) using oligo-dT or random hexamer primers according to the manufacturer's instructions.
  • Quantitative PCR was performed according to the manufacturer's instructions using a BioRad iCycler with approximately 50 ng cDNA per reaction containing 250 nM of each primer and 1 x SYBR green master mix (Bio-Rad) and analyzed by Bio-RAD thermocycler. The following conditions were used: Quantitative PCR reaction:
  • genes on the following list include 2 replicates each of 2 positive controls. Use 10 ul of each positive control per reaction. These are prepared such that the amount on the label is contained in 10 ul of solution.
  • normalized expression Input value average of gene/Input value average of internal control gene.
  • C-peptide ELISA C-peptide concentrations in conditioned medium were determined by ELISA with commercially available anti-C-peptide-coated plates (LINCO research) according to the manufacturer's recommendations.
  • Riboprobe synthesis Template plasmids were linearized with either Hind III (antisense) or BamH I (sense), and then purified (Qiagen Qiaquick spin columns), 1.5 ⁇ g of recovered DNA template was used in a synthesis reaction containing the corresponding RNA polymerase (Promega), nucleotides (DIG RNA labeling mix - 10 mM ATP, CTP, GTP (each), 6.5 mM UTP, 3.5 mM DIG-Il-UTP), 1 x Transcription buffer (1 x), and 25 Units RNAsin (Promega).
  • RNA probes were precipitated by the addition of 0.1 volumes of 4 M LiCl and 2.5 volumes of 100% ethanol and incubated at -20°C overnight. Samples were then centrifuged at 13,000 rpm for 30 min. at 4 0 C. The supernatant was discarded and the pellet was washed with 70% ethanol : 30% DEPC-H 2 O and re-centrifuged for 15 min. The supernatant was removed, and the pellet allowed to dry. Probes were typically resuspended in 50 ⁇ l of DEPC- H 2 O, aliquoted and stored at -80°C.
  • EBs were rehydrated in a descending series of methanol : PBT washes (75%, 50%, and 25% methanol). PBT is prepared from 1 x PBS-DEPC plus 0.1% Tween-20. EBs were incubated for 1 hr in 6% H 2 O 2 in PBT and then rinsed 3 x in PBT. EBs were then treated for 5 min. in 10 ⁇ g/ml proteinase K in PBT, washed in 2 mg/ml glycine in PBT (5 min.), followed by 2 additional PBT washes (5 min.
  • EBs were incubated in hybridization solution (50% deionized formamide (Ambion), 5 x SSC, 0.1% Tween- 20 (Sigma), 0.1% SDS (Sigma), 50 ⁇ g/ml heparin (Sigma), 50 ⁇ g/ml yeast tRNA, 60 mM citric acid in DEPC treated H 2 O) for at least two hours at 7O 0 C.
  • hybridization solution 50% deionized formamide (Ambion), 5 x SSC, 0.1% Tween- 20 (Sigma), 0.1% SDS (Sigma), 50 ⁇ g/ml heparin (Sigma), 50 ⁇ g/ml yeast tRNA, 60 mM citric acid in DEPC treated H 2 O) for at least two hours at 7O 0 C.
  • Hybridization solution was then replaced with fresh solution containing 50-100 ng DIG-labeled riboprobe and incubated on a rocking platform overnight at 7O 0 C.
  • EBs were washed for 5 min. in Solution I (50% formamide, 5 x SSC, 60 mM citric acid, and 1% SDS in DEPC-treated H 2 O) pre-warmed to 70 0 C.
  • EBs were washed twice more in solution I for 30 min. each at 7O 0 C, and once in solution I for 30 min. at 65°C.
  • EBs were then washed 3 x in Solution II (50% formamide, 2 x SSC, 24 mM citric acid, 0.2% SDS, and 0.1 % Tween-20 in DEPC-treated H 2 O) for 30 min. each at 65 0 C.
  • EBs were cooled to room temperature and washed 3 x (5 min. each) in maleic acid buffer (100 mM maleic acid (Sigma), 170 mM NaCl (Sigma), 0.1% Tween-20, and 2 niM levamisole, pH 7.5 with NaOH) (MAB).
  • MAB maleic acid buffer
  • MAB 170 mM NaCl
  • Tween-20 0.1%
  • Blocking solution was then replaced with fresh blocking solution containing pre- adsorbed alkaline phosphatase (AP)-conjugated anti-digoxygenin antibody (Roche) and incubated on a rocking platform overnight at 4 0 C. 2 ⁇ l antibody per ml was pre- adsorbed by incubation in MAB with 2% Boehringer Mannheim blocking reagent, 1% heat inactivated sheep serum, and 3 mg human EB acetone powder at 4°C for 90 min. and centrifuged for 10 min. at 4°C. EBs were washed 3 x (5 min. each) and 5 x (60-90 min. each) in MAB at room temperature and then incubated overnight in MAB at 4°C.
  • AP alkaline phosphatase
  • EBs were washed 3 x (10 min. each) in AP buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl 2 , 0.1% Tween-20, and 2 mM levamisole). EBs were then incubated in NTMT alkaline phosphatase staining buffer (AP buffer with 3.5 ⁇ l/ml NBT and 3.5 ⁇ l/ml BCIP) or alkaline phosphatase staining solution (BM Purple, Boehringer Mannheim) until the precipitation reaction was complete. Reaction was arrested with the addition of stop solution (2 mM EDTA in PBT).
  • AP buffer 100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl 2 , 0.1% Tween-20, and 2 mM levamisole.
  • NTMT alkaline phosphatase staining buffer AP buffer with 3.5 ⁇ l/ml NBT and 3.5

Abstract

La présente invention concerne des méthodes pour la différenciation dirigée de cellules souches embryonnaires le long de la lignée endodermique, notamment de la lignée pancréatique.
EP06719900A 2005-01-31 2006-01-30 Differenciation dirigee de cellules souches embryonnaires et utilisations associees Withdrawn EP1859026A2 (fr)

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